Bacterial vaccine components and uses thereof

ABSTRACT

Agents, compositions, methods and kits useful for the treatment and diagnosis of Staphylococcal intramammary infection are disclosed. The agents, compositions, methods and kits are derived from genes expressed during Staphylococcal intramammary infection, and more particularly genes SACOL0029, SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599, based on the gene nomenclature from the  Staphylococcus aureus  COL (SACOL) genome.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation application of U.S. application Ser.No. 14/513,987, now issued under U.S. Pat. No. 9,566,322, which is aDivisional application of U.S. application Ser. No. 13/583,054 filed onNov. 15, 2012, now issued under U.S. Pat. No. 8,889,150, which is aNational Entry application of PCT Application No. PCT/CA2011/050145filed on Mar. 17, 2011 and published in English under PCT Article 21(2),which itself claims benefit of U.S. Provisional Application Ser. No.61/314,670, filed on Mar. 17, 2010. All documents above are incorporatedherein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to novel vaccine targets and components.More specifically, the present invention is concerned with novelantigens which represent vaccine components, processes of manufacturingsame, methods using same, and methods of preventing and treatingmicrobial infections involving the administration of same.

REFERENCE TO SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named 14870_14-Sequence_listing, createdon Nov. 28, 2016 and having a size of ˜194 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Bovine mastitis is the most frequent and costly disease for dairyproducers and Staphylococcus aureus is considered to be thetransmittable bacterium that is the most often responsible for thedevelopment of the disease (Sears et al., 2003). Staphylococcalintramammary infections (IMI), which may lead to mastitis, are difficultto treat and frequent relapses are common (Sandholm et al., 1990).Bacterial susceptibility to antibiotics in vitro is a poor predictor oftherapeutic efficacy in chronically infected cows (Owens et al., 1997).Although infections that follow treatment of mastitis can be due tonewly acquired strains, they are often the result of the persistence ofthe original infective organism (Sandholm et al., 1990; Myllys et al.,1997). Existing therapies thus often fail to eliminate the infection andit would be highly desirable to find novel approaches to prevent ortreat staphylococcal IMI.

A lack of vaccine efficacy and protective ability has been noted forcommercially available S. aureus vaccines (Middleton, 2008). A number ofadditional Staphylococci vaccines and vaccine components have beendescribed and proposed. The use of milk or low-iron media as surrogatesystems for exploring S. aureus genes that are expressed during IMI donot fully replicate the actual mammalian host environment that may varyin nutrient composition, in interactions with host cells and in immuneresponse components, to name just a few differences. Hence, the S.aureus components currently proposed as vaccine are not necessarily thecomponents that are expressed during IMI at multiple points in time, bymultiple strains (including chronic strains) and in multiple hosts. Thusit would be highly desirable to identify S. aureus genes that areexpressed during IMI at multiple points in time, by multiple strains,and in multiple hosts, so that a selection of genes and gene-encodedproducts (e.g., proteins) can be used either alone or in combination forprotection against IMI and mastitis.

The present invention seeks to meet these and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

In an aspect, the present invention provides a method for preventingand/or treating Staphylococcal intramammary infection (IMI) in a mammal,said method comprising administrating to said mammal an effective amountof at least one agent, wherein said agent is: (a) a polypeptide encodedby a gene, wherein said gene is SACOL0029, SACOL0264, SACOL0442,SACOL0718, SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944,SACOL2144, SACOL2365 or SACOL2599, based on the gene nomenclature fromthe Staphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (b) a polypeptide encoded by a gene from a sameoperon as one of the genes of (a); (c) an immunogenic fragment of (a) or(b); (d) an immunogenic variant of any one of (a) to (c); (e) a nucleicacid encoding the polypeptide of any one of (a) to (d); or (f) anycombination of (a) to (e).

In another aspect, the present invention provides a use of an agent,wherein said agent is: (a) a polypeptide encoded by a gene, wherein saidgene is SACOL0029, SACOL0264, SACOL0442, SACOL0718, SACOL0720,SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144, SACOL2365 orSACOL2599 based on the gene nomenclature from the Staphylococcus aureusCOL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b)a polypeptide encoded by a gene from a same operon as one of the genesof (a); (c) an immunogenic fragment of (a) or (b); (d) an immunogenicvariant of any one of (a) to (c); (e) a nucleic acid encoding thepolypeptide of any one of (a) to (d); or (f) any combination of (a) to(e), for preventing and/or treating Staphylococcal intramammaryinfection (IMI) in a mammal.

In another aspect, the present invention provides a use of an agent,wherein said agent is: (a) a polypeptide encoded by a gene, wherein saidgene is SACOL0029, SACOL0264, SACOL0442, SACOL0718, SACOL0720,SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144, SACOL2365 orSACOL2599 based on the gene nomenclature from the Staphylococcus aureusCOL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b)a polypeptide encoded by a gene from a same operon as one of the genesof (a); (c) an immunogenic fragment of (a) or (b); (d) an immunogenicvariant of any one of (a) to (c); (e) a nucleic acid encoding thepolypeptide of any one of (a) to (d); or (f) any combination of (a) to(e), for the preparation of a medicament for preventing and/or treatingStaphylococcal intramammary infection (IMI) in a mammal.

In another aspect, the present invention provides a pharmaceuticalcomposition for preventing and/or treating Staphylococcal intramammaryinfection (IMI) in a mammal, said composition comprising: (a) at leastone agent, wherein said agent is (i) a polypeptide encoded by a gene,wherein said gene is SACOL0029, SACOL0264, SACOL0442, SACOL0718,SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144,SACOL2365 or SACOL2599 based on the gene nomenclature from theStaphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (ii) a polypeptide encoded by a gene from a sameoperon as one of the genes of (i); (iii) an immunogenic fragment of (i)or (ii); (iv) an immunogenic variant of any one of (i) to (iii); (v) anucleic acid encoding the polypeptide of any one of (i) to (iv); or (vi)any combination of (i) to (v). It may optionally comprise (b) apharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising: (a) at least one agent, wherein said agent is:(i) a polypeptide encoded by a gene, wherein said gene is SACOL0029,SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416,SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599, based on thegene nomenclature from the Staphylococcus aureus COL (SACOL) genome setforth in NCBI Reference Sequence NC_002951.2; (ii) a polypeptide encodedby a gene from a same operon as one of the genes of (a); (iii) animmunogenic fragment of (i) or (ii); (iv) an immunogenic variant of anyone of (i) to (iii); (v) a nucleic acid encoding the polypeptide of anyone of (i) to (iv); or (vi) any combination of (i) to (v); and (b) apharmaceutically acceptable excipient.

In another aspect, the present invention provides a kit for theprevention and/or treatment of Staphylococcal IMI, comprising (a) atleast one agent, wherein said agent is: (i) a polypeptide encoded by agene, wherein said gene is SACOL0029, SACOL0264, SACOL0442, SACOL0718,SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144,SACOL2365 or SACOL2599, based on the gene nomenclature from theStaphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (ii) a polypeptide encoded by a gene from a sameoperon as one of the genes of (a); (iii) an immunogenic fragment of (i)or (ii); (iv) an immunogenic variant of any one of (i) to (iii); (v) anucleic acid encoding the polypeptide of any one of (i) to (iv); or (vi)any combination of (i) to (v); and (b) instructions to use the kit forthe prevention and/or treatment of Staphylococcal IMI.

In another aspect, the present invention provides a method of diagnosingStaphylococcal IMI in a mammal, said method comprising: determining alevel of expression of at least one gene, wherein said gene isSACOL0029, SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353,SACOL1416, SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599, orthe level of activity of a polypeptide encoded by said one or moregenes, in a biological sample from said mammal; and comparing said levelof expression or activity to a reference level of expression oractivity; wherein a higher expression or activity in said biologicalsample relative to said reference expression or activity is indicativethat said mammal has staphylococcal IMI.

In another aspect, the present invention provides a kit for thediagnosis of Staphylococcal IMI, comprising (a) at least one ligand,wherein said at least one ligand binds to: (i) a polypeptide encoded bya gene, wherein said gene is SACOL0029, SACOL0264, SACOL0442, SACOL0718,SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144,SACOL2365 or SACOL2599, based on the gene nomenclature from theStaphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (ii) a polypeptide encoded by a gene from a sameoperon as one of the genes of (a); (iii) an immunogenic fragment of (i)or (ii); (iv) an immunogenic variant of any one of (i) to (iii); (v) anucleic acid encoding the polypeptide of any one of (i) to (iv); or (vi)any combination of (i) to (v); and (b) instructions to use the kit forthe diagnosis of Staphylococcal IMI.

In another aspect, the present invention provides a method forpreventing and/or treating Staphylococcal intramammary infection (IMI)in a mammal, said method comprising administrating to said mammal aneffective amount of at least one agent, wherein said agent is a liveattenuated form of Staphyloccocus aureus comprising a mutation in agene, wherein said gene is SACOL0029, SACOL0264, SACOL0442, SACOL0718,SACOL0720, SACOL1353, SACOL1416, SACOL1611, SACOL1944, SACOL2144,SACOL2365 or SACOL2599, based on the gene nomenclature from theStaphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2, and wherein the mutation is a deletion or aninsertion.

In another aspect, the present invention provides a use of an agent,wherein said agent is a live attenuated form of Staphyloccocus aureuscomprising a mutation in a gene, wherein said gene is SACOL0029,SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416,SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599, based on thegene nomenclature from the Staphylococcus aureus COL (SACOL) genome setforth in NCBI Reference Sequence NC_002951.2, and wherein the mutationis a deletion, an insertion or a substitution of one or morenucleotides, for preventing and/or treating Staphylococcal intramammaryinfection (IMI) in a mammal or for the preparation of a medicament forpreventing and/or treating Staphylococcal intramammary infection (IMI)in a mammal.

In another aspect, the present invention provides a pharmaceuticalcomposition for preventing and/or treating Staphylococcal intramammaryinfection (IMI) in a mammal, said composition comprising an agent,wherein said agent is a live attenuated form of Staphyloccocus aureuscomprising a mutation in a gene, wherein said gene is SACOL0029,SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416,SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599, based on thegene nomenclature from the Staphylococcus aureus COL (SACOL) genome setforth in NCBI Reference Sequence NC_002951.2, and wherein the mutationis a deletion, an insertion or a substitution of one or morenucleotides.

In another aspect, the present invention provides a kit for theprevention and/or treatment of Staphylococcal IMI, comprising at leastone agent, wherein said agent is a live attenuated form ofStaphyloccocus aureus comprising a mutation in a gene, wherein said geneis SACOL0029, SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353,SACOL1416, SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599,based on the gene nomenclature from the Staphylococcus aureus COL(SACOL) genome set forth in NCBI Reference Sequence NC_002951.2, andwherein the mutation is a deletion, an insertion or a substitution ofone or more nucleotides.

In an embodiment, the above-mentioned gene from the same operon as oneof the genes of (a) is SACOL0720, and wherein said one or more genes of(a) is SACOL0718.

In an embodiment, the above-mentioned one or more genes is SACOL0442,SACOL0718, SACOL0720 or any combination thereof. In a furtherembodiment, the above-mentioned one or more genes is SACOL0442,SACOL0720 or both.

In another embodiment, the above-mentioned methods, uses, pharmaceuticalcompositions or kits comprise a combination of agents. In a furtherembodiment, the above-mentioned combination of agents comprises: (i) afirst agent, wherein said first agent is (a) a polypeptide encoded bySACOL0442, (b) an immunogenic fragment of (a); (c) an immunogenicvariant of (a) or (b); (d) a nucleic acid encoding the polypeptide ofany one of (a) to (c); or (e) any combination of (a) to (d): and (ii) asecond agent, wherein said second agent is (a) a polypeptide encoded bySACOL0720, (b) an immunogenic fragment of (a); (c) an immunogenicvariant of (a) or (b); (d) a nucleic acid encoding the polypeptide ofany one of (a) to (c); or (e) any combination of (a) to (d).

In an embodiment, the above-mentioned gene is SACOL0442 and theimmunogenic fragment comprises one or more of the following amino acidsequences: TFGIYPKADASTQN (SEQ ID NO: 17), KDTINGKSNKSRNW (SEQ ID NO:18) or KDGGKYTLESHKELQ (SEQ ID NO: 19).

In another embodiment, the above-mentioned gene is SACOL0720 and theimmunogenic fragment comprises one or more of the following amino acidsequences: QFGFDLKHKKDALA (SEQ ID NO: 20), TIKDQQKANQLAS (SEQ ID NO:21), KDINKIYFMTDVDL (SEQ ID NO: 22) or DVDLGGPTFVLND (SEQ ID NO: 23).

In an embodiment, the above-mentioned Staphylococcal intramammaryinfection is caused by one or more Staphylococcus aureus strains.

In an embodiment, the above-mentioned methods, uses, pharmaceuticalcompositions or kits further comprise an adjuvant. In a furtherembodiment, the above-mentioned adjuvant is alum, Emulsigen™ D,cyclic-diguanosine-5′-monophosphate (c-di-GMP), polyphosphasine orpathogen-associated molecular patterns (PAMPS). In yet a furtherembodiment, the above-mentioned PAMPS is unmethylated dinucleotides(CpG) or microbial polysaccharides.

In an embodiment, the above-mentioned (i) agent, (ii) adjuvant, or both(i) and (ii) are comprised in a pharmaceutical composition.

In an embodiment, the above-mentioned pharmaceutical composition furthercomprises one or more pharmaceutically acceptable excipients.

In an embodiment, the above-mentioned mammal is a cow.

In an embodiment, the above-mentioned IMI is associated with bovinemastitis.

In an embodiment, the above-mentioned reference expression or activityis a level of expression or activity determined in a correspondingbiological sample from a mammal known to not having staphylococcal IMI.In another embodiment, the above-mentioned level of expression isdetermined by measuring the level of expression of a mRNA transcribedfrom said one or more genes. In another embodiment, said level ofexpression is determined by measuring the level of expression of apolypeptide encoded by said one or more genes.

In an embodiment, the above-mentioned biological sample is milk.

In an embodiment, the above-mentioned kit comprises a combination ofligands.

In an embodiment, the above-mentioned combination of ligands comprisesligands which bind to: (i) a first agent, wherein said first agent is(a) a polypeptide encoded by SACOL0442, (b) an immunogenic fragment of(a); (c) an immunogenic variant of (a) or (b); (d) a nucleic acidencoding the polypeptide of any one of (a) to (c); or (e) anycombination of (a) to (d): and (ii) a second agent, wherein said secondagent is (a) a polypeptide encoded by SACOL0720, (b) an immunogenicfragment of (a); (c) an immunogenic variant of (a) or (b); (d) a nucleicacid encoding the polypeptide of any one of (a) to (c); or (e) anycombination of (a) to (d).

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows the types of Staphylococcus aureus strains isolated fromcows: chronic and systematically isolated strains from cows withclinical symptoms. Chronic isolates were isolated from cows shedding agenetically identical S. aureus strain >55 days apart, between dry offand calving as illustrated (1st and 2^(nd) samples). Systematicallyisolated strains were taken at calving or in the lactation period fromcows shedding high somatic cell counts (SCC) in milk or having signs ofinflammation (mastitis). Somatic cells are leukocytes (white bloodcells). The SCC is an indicator of the quality of milk. The number ofsomatic cells increases in response to pathogenic bacteria like S.aureus.

FIG. 2 shows the genetic relatedness of the S. aureus isolates used inthe studies described herein as determined by comparative genomic DNAhybridization data obtained for 530 genes printed on DNA arrays.Underlined isolates are chronic strains. Unrelated reference strains andisolates randomly/systematically picked from bovine mastitis cases withclinical symptoms during lactation are shown for comparison.

FIG. 3 shows Q-PCR analyses reporting the relative level of geneexpression for indicators of virulence: hld (Agr-dependent exotoxinproduction), icaC (ica-dependent biofilm production) and overall biofilmproduction (measured by a spectrophotometric method with crystal violet)in S. aureus isolates grown in a cultivation medium in vitro. Chronicisolates #3 (black inverted triangle), #557 (black circle) and #1290(black diamond) are compared to a collection of systematically isolatedstrains from bovine mastitis with clinical signs (black squares, whereisolate SHY97-3906, a previously described strain isolated from atypical mastitis case with clinical signs, is represented as the opensquare). Q-PCR results are presented as fold-expression compared to thereference strain Newbould (ATCC 29740) and biofilm production isreported as a percentage of that produced by strain SHY97-3906. AllQ-PCR results are normalized using the level of expression of gyr.

FIG. 4 shows the description of the method used for isolating bacteriafrom mastitis milk samples. (A) Milk before first centrifugation with(Prot (+)) or without (Prot (−)) casein protease. (B) After firstcentrifugation. (C) After “RNA Protect” treatment and centrifugation(last step). A large bacterial pellet is recovered from milk treatedwith casein protease.

FIGS. 5A-5C show experimental infection profiles caused by chronicstrains #3, #557 and #1290 and by a strain isolated from a typicalmastitis case SHY97-3906 in cows reported as a function of bacterial(CFU) (left Y axis) or somatic cell counts (SCC) (right Y axis) over theinfection period. FIGS. 5A, 5B and 5C represent cows #313, cow #307 andcow #5325, respectively. The four different S. aureus strains used inthis study are represented as black bars and dots (SHY97-3906), whitebars and dots (chronic isolate #3), grey bars and dots (chronic isolate#557) and shaded bars and star-shaped dots (chronic isolate #1290). Cow#5325 was euthanized at day 15.

FIG. 6 shows a Venn diagram of the genes differentially expressed in thechronic strains taken all together (isolates #3, #557 and #1290) versusSHY97-3906 isolated from a typical mastitis case with clinical signs.

FIG. 7 shows a Venn diagram of the 43 genes found to be stronglyexpressed in microarray experiments using bacterial samples from cow#307 at day 8 (A) and day 10 (B) of infection, and in cow #5325 at day10 of infection (C). The number of bacterial samples in which the geneswere shown to be expressed is indicated in parenthesis and the genenames in bold characters were selected for Q-PCR analyses (see FIG. 8).

FIG. 8 shows quantitative PCR analyses of genes found to be stronglyexpressed by S. aureus collected from cow's IMI (cow). Gene expressionwas compared to that measured in S. aureus cultivated in vitro inMueller-Hinton broth supplemented with iron (broth+iron), iniron-restricted broth (broth−iron), and in freshly collectednon-mastitis milk in vitro (milk in vitro). All results are normalizedusing the level of expression of gyrB and are presented as Log 10 valuesof the relative expression ratios. The horizontal bar represents themedian ratio value of all samples. Significant differences between themedian and a ratio of zero (representing no change in the expression ofthe gene) are shown (*=P<0.05; **=P<0.01; ***=P<0.005; unpaired t-test).RNA samples from S. aureus grown in two different animals were analyzed:cow #5325 at day 10 of infection with isolates SHY97-3906 (∘), #3 (Δ),#557 (⋄), and #1290 (□), cow 307 at day 8 of infection (same symbolshapes, black symbols) and cow #307 at day 14 of infection (same symbolshapes, grey symbols). For the sample collected from strain #1290 in cow#5325 at day 10 of infection (□), the error bars are shown. Relativegene expression is shown for a capsular biosynthesis gene (capM), a geneof unknown function (SACOL2171), a transcriptional regulator of unknownfunction (SACOL2325), an ABC transporter of unknown function (SACOL0718)and a chromosomally encoded gene not previously characterized(SACOL0442).

FIG. 9 shows the organization of the SACOL0718-720 predicted operon inS. aureus strains COL, N315, RF122, USA300 and MSSA476. The two genesoverlap by 10 nucleotides. The arrow indicates the direction oftranscription. The genome position of the predicted −10 and -35 boxes ofthe promoter region is also indicated.

FIGS. 10A-10B show the growth kinetics of the S. aureus strain ATCC29213 and the isogenic three mutants ATCC 29213ΔSACOL0442a (Δ442a), ATCC29213ΔSACOL0442b (Δ442b) and ATCC29213 ΔSACOL0720 (Δ720). Mutants forgenes SACOL0442 and SACOL0720 were produced by gene replacement (Δ442a)or by intron insertion (Δ442b and Δ720). Prior to experimental bovineIMI, the relative growth of the parental strain and the mutants wasevaluated in vitro in freshly collected milk (FIG. 10A). In FIG. 10A,the mean CFU/ml (log 10) for the 4 strains is represented over timefollowing a small or large inoculum (left and right panels,respectively). FIG. 10B shows the mean bacterial counts recovered fromthe milk of eight (8) experimentally infected multiparous Holstein cowsin mid lactation as a function of time. Each of the 8 cows was infusedintra-mammary with the four S. aureus strains (ATCC 29213 and mutantsΔ442a, Δ442b, and Δ720) and the position of each strain in each of thefour mammary gland quarters alternated between the animals. Theinfections were carried out for 21 days. Milk of the infected quarterswas collected and the determination of viable bacterial counts wasperformed. Solid circles and open line represent growth of the parentstrain and the open symbols and solid lines the growth of the threemutants as indicated on the graph.

FIGS. 11A-11D show a nucleic acid (FIGS. 11A-C) and amino acid (FIG.11D) sequence alignment of SACOL0442 from various Staphylococcus aureusstrains (nucleic acid sequences: MW0345 (MW2) (SEQ ID NO: 24); SAS0347(MSSA476) (SEQ ID NO: 25); SACOL0442 (Col) (SEQ ID NO: 26);SAOUHSC_00354 (nctc8325) (SEQ ID NO: 27); NWMN_0362 (NEWMAN) (SEQ ID NO:28); SAUSA300_0370 (USA300-FPR3757) (SEQ ID NO: 29); SaurJH1_0429(JH1)(SEQ ID NO: 30); SAHV_0367 (Mu3) (SEQ ID NO: 31); SaurJH9_0419(JH9) (SEQ ID NO: 32); SAV0370 (Mu50) (SEQ ID NO: 33); SA0357 (N315)(SEQ ID NO: 34); SAB0321 (RF122)(SEQ ID NO: 35); and consensus (SEQ IDNO: 36); amino acid sequences: SACOL0442 (Col) (SEQ ID NO: 37);SAOUHSC_00354 (nctc8325) (SEQ ID NO: 38); NWMN_0362 (NEWMAN) (SEQ ID NO:39); SAUSA300_0370 (USA300-FPR3757) (SEQ ID NO: 40); SaurJH1_0429(JH1)(SEQ ID NO: 41); SAHV_0367 (Mu3) (SEQ ID NO: 42); SaurJH9_0419(JH9) (SEQ ID NO: 43); SAV0370 (Mu50) (SEQ ID NO: 44); SA0357 (N315)(SEQ ID NO: 45); SAS0347 (MSSA476) (SEQ ID NO: 46); SAB0321 (RF122)(SEQID NO: 47); and consensus (SEQ ID NO: 48). Alignments are based on thesequences available from multiple Staphylococcus aureus strains,including the bovine mastitis isolate RF122. The amino acid sequence ofMW0345 (MW2) (SEQ ID NO: 75) is not included in the alignment. Thecharacteristics of the compared strains are provided in FIG. 13. Undereach alignment, an asterisk (*) indicates a 100% match of nucleotide oramino acid between all strains compared; a double-dot (:) indicates thatconserved substitutions have been observed and a single dot (⋅) meansthat semi-conserved substitutions are observed.

FIGS. 12A-12K show a nucleic acid (FIGS. 12A-H) and amino acid (FIG.121-K) sequence alignment of SACOL0720 from various Staphylococcusaureus strains. (nucleic acid sequences: SaurJH1_0700 (JH1) (SEQ ID NO:49); SaurJH9_0685 (JH9) (SEQ ID NO: 50); SAHV_0659 (Mu3) (SEQ ID NO:51); SAV0662 (Mu50) (SEQ ID NO: 52); SA0617 (N315) (SEQ ID NO: 53);MW0624 (MW2) (SEQ ID NO:54); SAS0627 (MSSA476) (SEQ ID NO: 55);SACOL0720 (SEQ ID NO: 56); SAUSA300_0648 (USA300-FPR3757) (SEQ ID NO:57); SAOUHSC_00668 (NCTC8325) (SEQ ID NO: 58); NWMN_0631 (Newman) (SEQID NO: 59); SAB0611 (RF122) (SEQ ID NO: 60); consensus (SEQ ID NO: 61);amino acid sequence: SACOL0720 (SEQ ID NO: 62); SAUSA300_0648(USA300-FPR3757) (SEQ ID NO: 63); SAOUHSC_00668 (NCTC8325) (SEQ ID NO:64); NWMN_0631 (Newman) (SEQ ID NO: 65); SaurJH1_0700 (JH1) (SEQ ID NO:66); SaurJH9_0685 (JH9) (SEQ ID NO: 67); SAHV_0659 (Mu3) (SEQ ID NO:68); SAV0662 (Mu50) (SEQ ID NO: 69); SA0617 (N315) (SEQ ID NO: 70);MW0624 (MW2) (SEQ ID NO: 71); SAS0627 (MSSA476) (SEQ ID NO: 72); SAB0611(RF122) (SEQ ID NO: 73); consensus (SEQ ID NO: 74). Alignments are basedon the sequences available from multiple Staphylococcus aureus strains,including the bovine mastitis isolate RF122. The characteristics of thecompared strains are provided in FIG. 13. Under each alignment, anasterisk (*) indicates a 100% match of nucleotide or amino acid betweenall strains compared; a double-dot (:) indicates that conservedsubstitutions have been observed and a single dot (⋅) means thatsemi-conserved substitutions are observed.

FIG. 13 shows the characteristics of the Staphylococcus aureus strainswhose sequences are aligned at FIGS. 11A-D and 12A-K.

FIG. 14A-14C show the predicted cellular localization of proteinsSACOL0442 (FIG. 14A), SACOL0718 (FIG. 14B) and SACOL0720 (FIG. 14C), andFIG. 14D shows the predicted transmembrane helices of protein SACOL0720(SEQ ID NO: 62). In FIG. 14D, “I” and “i” represent intracellular aminoacids, “H” and “h” represent amino acids part of helix and are localizedin the membrane, “O” and “o” represent amino acids that areextracellular, i.e., localized outside the cytoplasmic membrane (capitalletters indicate a stronger prediction relative to lower case letters).The highlighted and boxed sequence is the longest extracellular sequenceof the protein that was used as a vaccine component in Example 7.Cellular localization was determined using the web site:http://psort.org/index.html (Gardy J. L. et al., Bioinformatics 200521(5):617-623; doi:10.1093/bioinformatics/bti057) and amino acidcomposition at the web site: http://www.expasy.ch/tools/protparam.html(Gasteiger E. et al., The Proteomics Protocols Handbook, Humana Press(2005), pp. 571-607). The localization of the transmembrane helix wasprovided by the server ExPASy™ proteomic server:http://www.enzim.hu/hmmtop/index.html (G. E Tusnády and I. Simon (2001),Bioinformatics 17, 849-850).

FIG. 15 shows total IgG titers in serums of mice vaccinated withSACOL0442 and SACOL0720. Antibody titers were determined by ELISA. Onegroup of animals (10 mice per group) received two injections of saline,one group received 2 injections of 100 μg of polypeptide SACOL0442, onegroup received 2 injections of 100 μg of polypeptide SACOL720, one groupreceived 2 injections of 100 μg of polypeptide SACOL1781, and one groupreceived 2 injections of 100 μg of each of all three polypeptidespremixed together (SACOL0442, SACOL0720, and SACOL1781). The 2injections were performed 3 weeks apart. Three weeks after the secondimmunization, mice were euthanized and blood collected for thedetermination of total IgG titers by ELISA. Each dot represents theserum titer of one mouse. Horizontal bars are the means for each group(dotted grey lines, antigens injected individually; solid black lines,antigens injected in combination).

FIG. 16 shows total IgG antibody titers in serums of dairy cowsvaccinated with SACOL0442 and SACOL0720. One group of animals (5 cowsper group) received two injections of saline, one group received 2injections of 300 μg of polypeptide SACOL0442, one group received 2injections of 300 μg of polypeptide SACOL720 and one group received 2injections of 300 μg of each of the two polypeptides premixed together(SACOL0442, SACOL0720). The 2 injections were performed 10 weeks apart.Blood was collected every two weeks for the determination of the totalIgG antibody titers by ELISA. Data represent the mean of each group.Solid lines and solid symbols represent total IgG antibody titer againstSACOL0720 and open lines and open symbols present total IgG antibodytiters against SACOL0442. Circles (solid for SACOL0720 and open forSACOL0442) represent the antibody titers for the group that receivedsaline. Triangles (solid for SACOL0720 and open for SACOL0442) representthe antibody titers for the two groups that received each one of the twopolypeptides. Squares (solid for SACOL0720 and open for SACOL0442)represent the antibody titers for the group that received bothpolypeptides.

FIGS. 17A-17D show IgG1 and IgG2 antibody titers at week 16, in serumsof dairy cows vaccinated with SACOL0442 and SACOL0720 (total IgG titersfrom the same samples at week 16 were shown in FIG. 16). As described inFIG. 16, one group of animals (5 cows per group) received two injectionsof saline, one group received 2 injections of 300 μg of polypeptideSACOL0442, one group received 2 injections of 300 μg of polypeptideSACOL720 and one group received 2 injections of 300 μg of each of thetwo polypeptides premixed together (SACOL0442, SACOL0720). The 2injections were performed 10 weeks apart. Blood was collected at week 16for the determination of IgG1 and IgG2 antibody titers by ELISA. FIGS.17A and B show IgG1 and IgG2 isotypes titers, respectively againstpolypeptide SACOL0442, FIGS. 17C and D show IgG1 and IgG2 isotypestiters, respectively against polypeptide SACOL0720. Each dot on thegraphs represents the serum titer of one cow (black squares, week 16;open circles, pre-immune titers before immunization). Horizontal barsare the medians for each group (solid lines, immune serums at week 16;open lines, pre-immune serums before immunizations).

FIG. 18 shows the amino acid (SEQ ID NO: 76) sequence of SACOL01781 fromS. aureus MW2 strain.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In a first aspect, the present invention provides a method forpreventing and/or treating Staphylococcal intramammary infection (IMI)in a mammal, said method comprising administrating to said mammal aneffective amount of an agent, wherein said agent is: (a) a polypeptideencoded by a gene, wherein said gene is SACOL0029, SACOL0100, SACOL0101,SACOL0105, SACOL0148, SACOL0154, SACOL0204, SACOL0205, SACOL0264,SACOL0442, SACOL0461, SACOL0608, SACOL0660, SACOL0688, SACOL0690,SACOL0704, SACOL0718, SACOL0720, SACOL0829, SACOL1054, SACOL1142,SACOL1145, SACOL1320, SACOL1353, SACOL1416, SACOL1611, SACOL1637,SACOL1680, SACOL1781, SACOL1812, SACOL1867, SACOL1912, SACOL1944,SACOL2092, SACOL2144, SACOL2169, SACOL2171, SACOL2321, SACOL2325,SACOL2342, SACOL2365, SACOL2379, SACOL2385 or SACOL2599, based on thegene nomenclature from the Staphylococcus aureus COL (SACOL) genome setforth in NCBI Reference Sequence NC_002951.2; (b) a polypeptide encodedby a gene from a same operon as one of the genes of (a); (c) animmunogenic fragment of (a) or (b); (d) an immunogenic variant of anyone of (a) to (c); (e) a nucleic acid encoding the polypeptide of anyone of (a) to (d); or (f) any combination of (a) to (e).

In another aspect, the present invention provides a use of an agent,wherein said agent is: (a) a polypeptide encoded by a gene, wherein saidgene is SACOL0029, SACOL0100, SACOL0101, SACOL0105, SACOL0148,SACOL0154, SACOL0204, SACOL0205, SACOL0264, SACOL0442, SACOL0461,SACOL0608, SACOL0660, SACOL0688, SACOL0690, SACOL0704, SACOL0718,SACOL0720, SACOL0829, SACOL1054, SACOL1142, SACOL1145, SACOL1320,SACOL1353, SACOL1416, SACOL1611, SACOL1637, SACOL1680, SACOL1781,SACOL1812, SACOL1867, SACOL1912, SACOL1944, SACOL2092, SACOL2144,SACOL2169, SACOL2171, SACOL2321, SACOL2325, SACOL2342, SACOL2365,SACOL2379, SACOL2385 or SACOL2599, based on the gene nomenclature fromthe Staphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (b) a polypeptide encoded by a gene from a sameoperon as one of the genes of (a); (c) an immunogenic fragment of (a) or(b); (d) an immunogenic variant of any of (a) to (c); (e) a nucleic acidencoding the polypeptide of any one of (a) to (d); or (f) anycombination of (a) to (e), for preventing and/or treating aStaphylococcal intramammary infection (IMI) in a mammal.

In another aspect, the present invention provides a use of an agent,wherein said agent is: (a) a polypeptide encoded by a gene, wherein saidagent is SACOL0029, SACOL0100, SACOL0101, SACOL0105, SACOL0148,SACOL0154, SACOL0204, SACOL0205, SACOL0264, SACOL0442, SACOL0461,SACOL0608, SACOL0660, SACOL0688, SACOL0690, SACOL0704, SACOL0718,SACOL0720, SACOL0829, SACOL1054, SACOL1142, SACOL1145, SACOL1320,SACOL1353, SACOL1416, SACOL1611, SACOL1637, SACOL1680, SACOL1781,SACOL1812, SACOL1867, SACOL1912, SACOL1944, SACOL2092, SACOL2144,SACOL2169, SACOL2171, SACOL2321, SACOL2325, SACOL2342, SACOL2365,SACOL2379, SACOL2385 or SACOL2599, based on the gene nomenclature fromthe Staphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2; (b) a polypeptide encoded by a gene from a sameoperon as one of the genes of (a); (c) an immunogenic fragment of (a) or(b); (d) an immunogenic variant of any one of (a) to (c); (e) a nucleicacid encoding the polypeptide of any one of (a) to (d); or (f) anycombination of (a) to (e), for the preparation of a medicament forpreventing and/or treating Staphylococcal intramammary infection (IMI)in a mammal.

In another aspect, the present invention provides a pharmaceuticalcomposition (e.g., a vaccine) for preventing and/or treatingStaphylococcal intramammary infection (IMI) in a mammal, saidcomposition comprising an agent, wherein said agent is: (a) apolypeptide encoded by a gene, wherein said gene is SACOL0029,SACOL0100, SACOL0101, SACOL0105, SACOL0148, SACOL0154, SACOL0204,SACOL0205, SACOL0264, SACOL0442, SACOL0461, SACOL0608, SACOL0660,SACOL0688, SACOL0690, SACOL0704, SACOL0718, SACOL0720, SACOL0829,SACOL1054, SACOL1142, SACOL1145, SACOL1320, SACOL1353, SACOL1416,SACOL1611, SACOL1637, SACOL1680, SACOL1781, SACOL1812, SACOL1867,SACOL1912, SACOL1944, SACOL2092, SACOL2144, SACOL2169, SACOL2171,SACOL2321, SACOL2325, SACOL2342, SACOL2365, SACOL2379, SACOL2385 orSACOL2599, based on the gene nomenclature from the Staphylococcus aureusCOL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b)a polypeptide encoded by a gene from a same operon as one of the genesof (a); (c) an immunogenic fragment of (a) or (b); (d) an immunogenicvariant of any one of (a) to (c); (e) a nucleic acid encoding thepolypeptide of any one of (a) to (d); or (f) any combination of (a) to(e), and optionally one or more pharmaceutically acceptableexcipients/carriers.

The Genbank accession numbers for the above-mentioned S. aureus genesand encoded polypeptides are depicted in Table I below:

TABLE I Genbank accession numbers for the IMI-associated S. aureus genesand encoded polypeptides described herein. Gene name GenBank Gene ID No.GenBank protein No. SACOL0029 3236748 YP_184940.1 (SEQ ID NO: 78) (SEQID NO: 77) SACOL0100 3236858 YP_185004.1 SACOL0101 3236840 YP_185005.1SACOL0105 3236844 YP_185009.1 SACOL0148 3236734 YP_185048.1 SACOL01543238707 YP_185054.1 SACOL0204 3236774 YP_185103.1 SACOL0205 3236775YP_185104.1 SACOL0264 3236683 YP_185159.1 (SEQ ID NO: 80) (SEQ ID NO:79) SACOL0442 3236485 YP_185332.1 (SEQ ID NO: 37) (SEQ ID NO: 81)SACOL0461 3236475 YP_185351.1 SACOL0608 3236353 YP_185493.1 SACOL06603238251 YP_185544.1 SACOL0688 3236721 YP_185570.1 SACOL0690 3236723YP_185572.1 SACOL0704 3236241 YP_185586.1 ISACOL0718 3236599 YP_185600.1(SEQ ID NO: 83) (SEQ ID NO: 82) SACOL0720 3236600 YP_185601.1 (SEQ IDNO: 62) (SEQ ID NO: 84) SACOL0829 3238649 YP_185703.1 SACOL1054 3236163YP_185919.1 SACOL1142 3236098 YP_186005.1 SACOL1145 3237661 YP_186008.1SACOL1320 3236394 YP_186175.1 SACOL1353 3236077 YP_186206.1 (SEQ ID NO:86) (SEQ ID NO: 85) SACOL1416 3236563 YP_186268.1 (SEQ ID NO: 88) (SEQID NO: 87) SACOL1611 3236575 YP_186451.1 (SEQ ID NO: 90) (SEQ ID NO: 89)SACOL1637 3238018 YP_186477.1 SACOL1680 3238476 YP_186520.1 SACOL17813236594 YP_186614.1 SACOL1812 3238705 YP_186645.1 SACOL1867 3236101YP_186695.1 (SEQ ID NO: 99) (SEQ ID NO: 100) SACOL1912 3236086YP_186737.1 SACOL1944 3237515 YP_186769.1 (SEQ ID NO: 92) (SEQ ID NO:91) SACOL2092 3238693 YP_186907.1 SACOL2144 3237436 YP_186957.1 (SEQ IDNO: 94) (SEQ ID NO: 93) SACOL2169 3237416 YP_186981.1 SACOL2171 3237418YP_186983.1 SACOL2321 3238070 YP_187128.1 SACOL2325 3238483 YP_187132.1SACOL2342 3235997 YP_187148.1 SACOL2365 3238203 YP_187170.1 (SEQ ID NO:96) (SEQ ID NO: 95) SACOL2379 3237628 YP_187183.1 SACOL2385 3238646YP_187189.1 SACOL2599 3237186 YP_187390.1 (SEQ ID NO: 98) (SEQ ID NO:97)

In an embodiment, the above-mentioned gene is SACOL0029, SACOL0264,SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416, SACOL1611,SACOL1944, SACOL2144, SACOL2365 or SACOL2599.

As used herein, the term “vaccine” refers to any compound/agent(“vaccine component”), or combinations thereof, capable ofinducing/eliciting an immune response in a host and which permits totreat and/or prevent an infection and/or a disease. Therefore,non-limiting examples of such agent include proteins, polypeptides,protein/polypeptide fragments, immunogens, antigens, peptide epitopes,epitopes, mixtures of proteins, peptides or epitopes as well as nucleicacids, genes or portions of genes (encoding a polypeptide or protein ofinterest or a fragment thereof) added separately or in a contiguoussequence such as in nucleic acid vaccines, and the like.

An immunogenic fragment of a protein/polypeptide is defined as a part ofa protein/polypeptide which is capable of inducing/eliciting an immuneresponse in a host. In an embodiment, the immunogenic fragment iscapable of eliciting the same immune response in kind, albeit notnecessarily in amount, as the protein/polypeptide. An immunogenicfragment of a protein/polypeptide preferably comprises one or moreepitopes of said protein/polypeptide. An epitope of aprotein/polypeptide is defined as a fragment of said protein/polypeptideof at least about 4 or 5 amino acids in length, capable of eliciting aspecific antibody and/or an immune cell (e.g., a T cell or B cell)bearing a receptor capable of specifically binding said epitope. Twodifferent kinds of epitopes exist: linear epitopes and conformationalepitopes. A linear epitope comprises a stretch of consecutive aminoacids. A conformational epitope is typically formed by several stretchesof consecutive amino acids that are folded in position and together forman epitope in a properly folded protein. An immunogenic fragment as usedherein refers to either one, or both, of said types of epitopes. In anembodiment, the immunogenic fragment of a protein/polypeptide comprisesat least 4 or 5 amino acid residues. In a further embodiment, theimmunogenic fragment comprises at least 6, 7, 8, 9, 10, 13, 14, 15, 20,25, 30, 50 or 100 consecutive amino acids of the nativeprotein/polypeptide.

As will be understood by the person of ordinary skill, agents(proteins/polypeptides, fragments thereof) having non-naturallyoccurring modifications (e.g., immunogenic variants) and which arecapable of inducing an immune response specific for the unmodified agent(e.g., capable of inducing the production of antibodies capable ofrecognizing the unmodified agent) are also within the scope of the term“vaccine component”. For example, the vaccine components of the presentinvention can be modified to enhance their activity, stability, and/orbioavailability, and/or to reduce their toxicity. Conservative aminoacid substitutions may be made, like for example replacement of an aminoacid comprising an acidic side chain by another amino acid comprising anacidic side chain, replacement of a bulky amino acid by another bulkyamino acid, replacement of an amino acid comprising a basic side chainby another amino acid comprising a basic side chain, and the like. Aperson skilled in the art is well able to generate variants of aprotein/polypeptide. This is for instance done through screening of apeptide library or by peptide changing programs. An immunogenic variantaccording to the invention has essentially the same immunogenicproperties of said protein in kind, not necessarily in amount. Animmunogenic variant of a protein/polypeptide of the invention may forinstance comprise a fusion protein and/or chimeric protein. For example,the biological function of protein SACOL0442 identified herein ispredicted to be an exotoxin, enterotoxin or superantigen and it couldpotentially interfere with the mammalian immune system and antibodyproduction, and/or show some toxicity in the host. Although suchinterference was not observed when the SACOL0442 polypeptide was used incombination with for example SACOL0720 during immunization (FIG. 15), itmay be useful to modify the protein or polypeptide used for vaccinationso that the biological activity of the exotoxin is decreased. For such apurpose, it is possible to inactivate the exotoxin with chemicals (e.g.,formaldehyde). It is also possible to use molecular biology techniquesto delete or mutate the putative region(s) involved in exotoxin activitywithout loosing immunogenicity (Chang et al., 2008). Another example isthe conjugation or mixture of amino acid-based components with nucleicacids (e.g., genes or portions of genes added separately or in acontiguous sequence) carbohydrates such as those found in microbialpolysaccharide capsules or biofilms.

In an embodiment, the above-mentioned polypeptide is a polypeptidenormally secreted or expressed at the surface of the bacteria (e.g.,Staphylococcus aureus).

In another embodiment, the above-mentioned polypeptide, or a polypeptidesubstantially identical to said polypeptide, is expressed in at leasttwo different strains of Staphylococcus aureus. Substantially identicalas used herein refers to polypeptides having at least 60% of similarity,in embodiments at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of similarity in their amino acidsequences. In further embodiments, the polypeptides have at least 60%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%of identity in their amino acid sequences.

In an embodiment, the above-mentioned immunogenic fragment comprises asequence that is conserved (i.e. identical) in at least two differentstrains of Staphylococcus aureus. In further embodiments, theabove-mentioned immunogenic fragment comprises a sequence that isconserved (i.e. identical) in at least 3, 4, 5, 6, 7, 8, 9 or 10different strains of Staphylococcus aureus. In another embodiment, theabove-mentioned strains of Staphylococcus aureus are COL, RF122, NCTC8325, JH1, JH9, Newman, Mu3, Mu50, USA300-FPR3757, N315, MW2 or MSSA476.In an embodiment, the above-mentioned strains of Staphylococcus aureusis associated with bovine mastitis (e.g., RF122).

The similarity and identity between amino acid or nucleotide sequencescan be determined by comparing each position in the aligned sequences.Optimal alignment of sequences for comparisons of similarity and/oridentity may be conducted using a variety of algorithms, for exampleusing a multiple sequence alignment program/software well known in theart such as ClustalW™, SAGA™, UGENE™ or T-coffee™. Examples of multiplesequence alignments are described in the examples below and depicted inFIGS. 11A-D and FIGS. 12A-K.

Also within the context of the present invention is the in vivoadministration of a nucleic acid of the invention to a mammal so thatone or more proteins/polypeptides (or a fragment thereof) of interestis/are expressed in the mammal (e.g., nucleic acid vaccine, DNA or RNAvaccine).

The nucleic acid of the present invention preferably comprises anucleotide sequence that encodes one or more proteins/polypeptides notedabove (or fragments thereof) operably linked to regulatory elementsneeded for gene expression, such as a promoter, an initiation codon, astop codon, enhancers, and a polyadenylation signal. Regulatory elementsare preferably selected that are operable in the species to which theyare to be administered.

The nucleic acid of the present vaccine can be “naked” DNA or can beoperably incorporated in a vector. Nucleic acids may be delivered tocells in vivo using methods well known in the art such as directinjection of DNA, receptor-mediated DNA uptake, viral-mediatedtransfection or non-viral transfection and lipid-based transfection, allof which may involve the use of vectors. Direct injection has been usedto introduce naked DNA into cells in vivo (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). Adelivery apparatus (e.g., a “gene gun”) for injecting DNA into cells invivo may be used. Such an apparatus may be commercially available (e.g.,from BioRad). Naked DNA may also be introduced into cells by complexingthe DNA to a cation, such as polylysine, which is coupled to a ligandfor a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988)J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complexto the receptor may facilitate uptake of the DNA by receptor-mediatedendocytosis. A DNA-ligand complex linked to adenovirus capsids whichdisrupt endosomes, thereby releasing material into the cytoplasm, may beused to avoid degradation of the complex by intracellular lysosomes (seefor example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850;Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Useful delivery vectors include biodegradable microcapsules,immuno-stimulating complexes (ISCOMs) or liposomes, and geneticallyengineered attenuated live vectors such as viruses or bacteria. Examplesof suitable attenuated live bacterial vectors include Salmonellatyphimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus,Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae,Campylobacter, or any other suitable bacterial vector, as is known inthe art. Methods of transforming live bacterial vectors with anexogenous DNA construct are well described in the art. See, for example,Joseph Sambrook and David W. Russell, Molecular Cloning, A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001).

Preferred viral vectors include Bacteriophages, Herpes virus,Adenovirus, Polio virus, Vaccinia virus, defective retroviruses,adeno-associated virus (AAV) and Avipox. Methods of transforming viralvector with an exogenous DNA construct are also well described in theart. See Sambrook and Russell, above.

Liposome vectors are unilamellar or multilamellar vesicles, having amembrane portion formed of lipophilic material and an interior aqueousportion. The aqueous portion is used in the present invention to containthe polynucleotide material to be delivered to the target cell. It isgenerally preferred that the liposome forming materials have a cationicgroup, such as a quaternary ammonium group, and one or more lipophilicgroups, such as saturated or unsaturated alkyl groups having about 6 toabout 30 carbon atoms. One group of suitable materials is described inEuropean Patent Publication No. 0187702, and further discussed in U.S.Pat. No. 6,228,844 to Wolff et al., the pertinent disclosures of whichare incorporated by reference. Many other suitable liposome-formingcationic lipid compounds are described in the literature. See, e.g., L.Stamatatos, et al., Biochemistry 27:3917 3925 (1988); and H. Eibl, etal., Biophysical Chemistry 10:261 271 (1979). Alternatively, amicrosphere such as a polylactide-coglycolide biodegradable microspherecan be utilized. A nucleic acid construct is encapsulated or otherwisecomplexed with the liposome or microsphere for delivery of the nucleicacid to a tissue, as is known in the art.

Alternatively, the nucleic acid (e.g., DNA or RNA) may be incorporatedin a cell in vitro or ex vivo by transfection or transformation, and thetransfected or transformed cell (e.g., an immune cell such as adendritic cell), which expresses the protein or polypeptide of interest(or a fragment thereof), may be administered to the host. Followingadministration, the cell will express the protein or polypeptide ofinterest (or a fragment thereof) in the host, which will in turn lead tothe induction of an immune response directed against the protein,polypeptide or fragment thereof.

Also encompassed by the methods, uses, pharmaceutical compositions andkits of the present invention is passive immunization, which is theinjection of antibodies or antiserum, previously generated against thepathogen, in order to protect or cure a recipient animal of an infectionor future infection. Protection fades over the course of a few weeksduring which time the active immunization with protein and/or DNA (asdescribed above) will have time to generate a lasting protectiveresponse. Serum for passive immunization can be generated byimmunization of donor animals using the S. aureus antigens (proteins,polypeptides or nucleic acids), as described above. This serum, whichcontains antibodies against the antigens, can be used immediately orstored under appropriate conditions. It can be used to combat acuteinfections (IMI) or as a prophylactic (Tuchscherr et al., 2008). Use ofantibodies or serums in a passive immunization can be combined withother agents such as an antibiotic to increase the cure rate of aninfection currently in progress or to increase protection against animminent infection.

Also encompassed by the methods, uses, pharmaceutical compositions andkits of the present invention is immunization with the Staphylococcusaureus bacteria in attenuated live or inactivated form (e.g., S. aureushaving at least one of the genes of the present invention mutated (e.g.,Δ442a, Δ442b and Δ720 of SACOL442 and SACOL720, as described in Example6). Mutation as used herein includes a substitution, a deletion and/oran insertion of one or more nucleotides that prevents expression of thepolypeptide encoded by a gene of the present invention or that preventsexpression of a functional polypeptide. In a preferred embodiment, themutation prevents expression of the polypeptide (e.g., Δ442a, Δ442b andΔ720 of SACOL442 and SACOL720, as described in Example 6). In anotherspecific embodiment, the mutation is a deletion or an insertion. It isexpected that a mutated strain of S. aureus having a mutation at anyposition of one of the genes of the present invention that preventsexpression of the polypeptide can be used as an attenuated live vaccinein accordance with the present invention. Attenuated live vaccines, i.e.vaccines comprising the bacterium according to the invention in a liveattenuated form, have the advantage over inactivated vaccines that theybest mimic the natural way of infection. In addition, their replicatingabilities allow vaccination with low amounts of bacteria; their numberwill automatically increase until it reaches the trigger level of theimmune system. From that moment on, the immune system will be triggeredand will finally eliminate the bacteria. A minor disadvantage of the useof live attenuated bacteria however might be that inherently there is acertain level of virulence left. This need not be a real disadvantage aslong as the level of virulence is acceptable, i.e. as long as thevaccine at least descreases the mammal IMI symptoms. Of course, thelower the rest virulence of the live attenuated vaccine is, the lessinfluence the vaccination has on weight gain during/after vaccination.

The components identified in accordance with the teachings of thepresent invention have a prophylactic and/or therapeutic value such asthey can be used to raise an immune response to prevent and/or combatdiseases or conditions, and more particularly diseases or conditionsrelated to microbial infections.

The terms “treat/treating/treatment” and “prevent/preventing/prevention”as used herein, refers to eliciting the desired biological response,i.e., a therapeutic and prophylactic effect, respectively. In accordancewith the subject invention, the therapeutic effect comprises one or moreof a decrease/reduction in the severity of the disease (e.g., areduction or inhibition of infection), a decrease/reduction in symptomsand disease-related effects, an amelioration of symptoms anddisease-related effects, and an increased survival time of the affectedhost animal, following administration of the at least one agent (or of acomposition comprising the agent). In accordance with the invention, aprophylactic effect may comprise a complete or partialavoidance/inhibition or a delay of infection, and an increased survivaltime of the affected host animal, following administration of the atleast one agent (or of a composition comprising the agent).

As used herein, the term “pharmaceutically acceptable” refers to vaccinecomponents (e.g., excipients, carriers) and compositions that arephysiologically tolerable and do not typically produce an allergic orsimilar untoward reaction, such as gastric upset, dizziness and thelike, when administered to a subject. Preferably, as used herein, theterm “pharmaceutically acceptable” means approved by regulatory agencyof the federal or state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and inhumans. The term “excipient” refers to a diluent, carrier, or vehiclewith which the vaccine components of the present invention may beadministered. Sterile water or aqueous saline solutions and aqueousdextrose and glycerol solutions may be employed as carriers,particularly for injectable solutions.

In an embodiment, the agent of the present invention is administered incombination with an adjuvant or immunostimulant. Suitable adjuvant orimmunostimulant that may improve the efficacy of components to raise animmune response include but is not limited to oils (e.g., mineral oils,emulsified oil such as EMULSIGEN™-D), metallic salts (e.g., alum,aluminum hydroxide or aluminum phosphate), natural and artificialmicrobial components (e.g., bacterial liposaccharides, Freund'sadjuvants, muramyl dipeptide (MDP), cyclic-diguanosine-5′-monophosphate(c-di-GMP), pathogen-associated molecular patterns (PAMPS)), plantcomponents (e.g., Quil A), and/or one or more substances that have acarrier effect (e.g., bentonite, latex particles, liposomes, ISCOM™ andpolyphosphazine (PCPP) copolymers). Immunization with syntheticnanoparticles (such as those made from a biodegradable synthetic polymerlike poly(D,L-lacticco-glycolic acid)) containing antigens plus ligandsthat signal through TLR to stimulate proinflammatory cytokines is alsopossible (Kasturi et al, 2011).

Vaccine components of the invention may be administered in apharmaceutical composition. Pharmaceutical compositions may beadministered in unit dosage form. Any appropriate route ofadministration may be employed, for example, parenteral, subcutaneous,intramuscular, intramammary, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraarticular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, or oraladministration. Examples of specific routes of administration includeparenteral, e.g., intravenous, intradermal, subcutaneous, intramammary;oral (e.g., inhalation); transdermal (topical); transmucosal, and rectaladministration.

Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer such vaccine components withor without adjuvants to subjects. Methods well known in the art formaking pharmaceutical compositions and formulations are found in, forexample, Remington: The Science and Practice of Pharmacy, (20th ed.) ed.A. R. Gennaro A R., 2000, Lippincott: Philadelphia. Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol,miglyol, oils of vegetable origin, or hydrogenated napthalenes.Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Other potentially usefulparenteral delivery systems for compounds of the invention includeethylenevinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation orintramammary injection may contain excipients, or example, lactose, ormay be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, miglyol, glycocholate and deoxycholate,or may be oily solutions (e.g., paraffin oil) for administration in theform of nasal drops, or as a gel.

Therapeutic formulations may be in the form of liquid solutions orsuspension; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols. Solutions or suspensions used forparenteral, intradermal, intramammary or subcutaneous application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils (e.g., paraffin oil),polyethylene glycols, glycerine, propylene glycol, miglyol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; reducingagents such dithiothreitol, buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. The pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide. The parenteral preparation canbe enclosed in ampoules, disposable syringes or multiple dose vials madeof glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous or intramammary administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor™ ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS).

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets or feed. For the purpose of oral vaccine administration, theactive components can be incorporated with excipients and used in theform of tablets, troches, capsules or in feed. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel™, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the vaccine components are deliveredin the form of an aerosol spray from pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

Liposomal suspensions (including liposomes targeted to specific celltypes) can also be used as pharmaceutically acceptable carriers.

The pharmaceutical compositions may also contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts for the variation of osmoticpressure, buffers, coating agents or antioxidants. They may also containother therapeutically valuable agents.

Intravenous, intramuscular, intramammary or oral administration is apreferred form of use. The dosages in which the components of thepresent invention are administered in effective amounts depend on thenature of the specific active ingredient, the host and the requirementsof the subject and the mode of application. In general, an amount ofabout 0.01 mg-500 mg per dose, come into consideration.

Toxicity or efficacy of vaccine components to elicit an immune responsecan be determined by standard procedures in cell cultures orexperimental animals. The dose ratio between toxic and immunestimulatory effects can be measured. Components that exhibit largeratios are preferred. While components that exhibit toxic side effectsmay be used, care should be taken to design a delivery system in orderto minimize potential damage to cells and, thereby, reduce side effects.

Data obtained from cell culture assays and laboratory animal studies canbe used in formulating a range of dosage for use in large animals andhumans. The dosage of such components lies preferably within a range ofadministered concentrations that include efficacy with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively raise an immune response in asubject. Moreover, the therapeutically effective amount of a componentof the present invention may require a series of doses.

The present invention also encompasses kits comprising the components ofthe present invention. For example, the kit can comprise one or morecomponents. The components can be packaged in a suitable container anddevice for administration. The kit can further comprise instructions forusing the kit.

The present invention also provides a method of diagnosingStaphylococcal IMI in a mammal, said method comprising: determining alevel of expression of at least one gene, wherein said gene isSACOL0029, SACOL0100, SACOL0101, SACOL0105, SACOL0148, SACOL0154,SACOL0204, SACOL0205, SACOL0264, SACOL0442, SACOL0461, SACOL0608,SACOL0660, SACOL0688, SACOL0690, SACOL0704, SACOL0718, SACOL0720,SACOL0829, SACOL1054, SACOL1142, SACOL1145, SACOL1320, SACOL1353,SACOL1416, SACOL1611, SACOL1637, SACOL1680, SACOL1781, SACOL1812,SACOL1867, SACOL1912, SACOL1944, SACOL2092, SACOL2144, SACOL2169,SACOL2171, SACOL2321, SACOL2325, SACOL2342, SACOL2365, SACOL2379,SACOL2385 or SACOL2599, based on the gene nomenclature from theStaphylococcus aureus COL (SACOL) genome set forth in NCBI ReferenceSequence NC_002951.2, or the level of activity of a polypeptide encodedby said one or more genes (at least one gene), in a biological samplefrom said mammal; and comparing said level of expression or activity toa reference level of expression or activity; wherein a higher expressionor activity in said biological sample relative to said referenceexpression or activity is indicative that said mammal has staphylococcalIMI.

In an embodiment, the above-mentioned reference expression or activityis a level of expression or activity determined in a correspondingbiological sample from a mammal known to not having staphylococcal IMI.Such reference expression or activity may be an expression or activitycorresponding to an average or median expression or activity calculatedbased on measurements made in several subjects not suffering from thecondition (e.g., known to not having staphylococcal IMI). The referenceexpression or activity may be adjusted or normalized for age, gender,race, or other parameters.

In an embodiment, the above-mentioned at least one gene is SACOL0029,SACOL0264, SACOL0442, SACOL0718, SACOL0720, SACOL1353, SACOL1416,SACOL1611, SACOL1944, SACOL2144, SACOL2365 or SACOL2599.

“Sample” or “biological sample” refers to any solid or liquid sampleisolated from a live being. In a particular embodiment, it refers to anysolid (e.g., tissue sample) or liquid sample isolated from a mammal,such as milk, a biopsy material (e.g., solid tissue sample), blood(e.g., plasma, serum or whole blood), saliva, synovial fluid, urine,amniotic fluid and cerebrospinal fluid. Such sample may be, for example,fresh, fixed (e.g., formalin-, alcohol- or acetone-fixed),paraffin-embedded or frozen prior to analysis of the infectious agent'sexpression level.

In an embodiment, the above-mentioned biological sample is milk.

In an embodiment, the above-mentioned mammal is a cow.

In an embodiment, the above-mentioned level of expression is determinedby measuring the level of expression of a polypeptide/protein encoded bysaid one or more genes. Methods to measure the amount/level of selectedpolypeptides/proteins of this invention (one or more of the polypeptidesnoted above) are well known in the art. Protein/polypeptide levels maybe detected either directly using affinity reagents, such as an antibodyor a fragment thereof (for methods, see for example Harlow, E. and Lane,D (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), or a ligand (natural or synthetic)which binds the protein. Protein/polypeptide levels may be detectedbased on other properties, for example by measurement of the protein'sactivity, which may entail enzymatic activity to produce a detectableproduct (e.g., with altered spectroscopic properties) or a detectablephenotype (e.g., alterations in cell growth/function).

Examples of methods to measure the amount/level of selectedproteins/polypeptides include, but are not limited to: Western blot,immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay(RIA), immunoprecipitation, surface plasmon resonance,chemiluminescence, fluorescent polarization, phosphorescence,immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,microcytometry, microarray, microscopy, flow cytometry, and assays basedon a property of the protein including but not limited to DNA binding,ligand binding, interaction with other protein partners or enzymaticactivity.

In an embodiment, the amount of the polypeptide/protein within themethods of the present invention is detected using antibodies that aredirected specifically against the polypeptide/protein. The term“antibody” as used herein encompasses monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments, so long as they exhibit the desired biologicalactivity or specificity. “Antibody fragments” comprise a portion of afull-length antibody, generally the antigen binding or variable regionthereof. Interactions between antibodies and a target polypeptide aredetected by radiometric, colorimetric, or fluorometric means. Detectionof antigen-antibody complexes may be accomplished by addition of asecondary antibody that is coupled to a detectable tag, such as forexample, an enzyme, fluorophore, or chromophore.

Methods for making antibodies are well known in the art. Polyclonalantibodies can be prepared by immunizing a suitable subject (e.g.,rabbit, goat, mouse, or other mammal) with the polypeptide/protein ofinterest or a fragment thereof as an immunogen. A polypeptide/protein“fragment” “portion” or “segment” is a stretch of amino acid residues ofat least about 5, 7, 10, 14, 15, 20, 21 or more amino acids of thepolypeptide noted above. The antibody titer in the immunized subject canbe monitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized exosomal markerpolypeptide or a fragment thereof. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the animal, usually amouse, and can be used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256: 495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4: 72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology, John Wiley & Sons, Inc., New York,N.Y.).

Alternatively to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with a polypeptide or a fragment thereof tothereby isolate immunoglobulin library members that bind thepolypeptide. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System™, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612).

Furthermore, antibodies directed against one or more of thepolypeptides/proteins described herein may be obtained from commercialsources.

The use of immobilized antibodies specific for the polypeptides/proteinsis also contemplated by the present invention and is well known by oneof ordinary skill in the art. The antibodies could be immobilized onto avariety of solid supports, such as magnetic or chromatographic matrixparticles, the surface of an assay place (such as microtiter wells),pieces of a solid substrate material (such as plastic, nylon, paper),and the like. An assay strip could be prepared by coating the antibodyor a plurality of antibodies in an array on solid support. This stripcould then be dipped into the test sample and then processed quicklythrough washes and detection steps to generate a measurable signal, suchas a colored spot.

The analysis of a plurality (2 or more) of polypeptides/proteins may becarried out separately or simultaneously with one test sample. Severalpolypeptides/proteins may be combined into one test for efficientprocessing of a multiple of samples.

The analysis of polypeptides/proteins could be carried out in a varietyof physical formats as well. For example, the use of microtiter platesor automation could be used to facilitate the processing of largenumbers of test samples. Alternatively, single sample formats could bedeveloped to facilitate immediate treatment and diagnosis in a timelyfashion. Particularly useful physical formats comprise surfaces having aplurality of discrete, addressable locations for the detection of aplurality of different analytes. Such formats include proteinmicroarrays, or “protein chips” (see, e.g., Ng and Ilag, J. Cell Mol.Med. 6: 329-340, 2002) and capillary devices.

In an embodiment, the above-mentioned level of expression is determinedby measuring the level of expression of a mRNA transcribed from said oneor more genes.

Methods to determine nucleic acid (mRNA) levels are known in the art,and include for example polymerase chain reaction (PCR), reversetranscriptase-PCR (RT-PCR), SAGE, quantitative PCR (q-PCR), Southernblot, Northern blot, sequence analysis, microarray analysis, detectionof a reporter gene, or other DNA/RNA hybridization platforms. For RNAexpression, preferred methods include, but are not limited to:extraction of cellular mRNA and Northern blotting using labeled probesthat hybridize to transcripts encoding all or part of one or more of thenucleic acids encoding the protein/polypeptide of this invention;amplification of mRNA expressed from one or more of the nucleic acidsencoding the proteins/polypeptides of this invention using specificprimers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), andreverse transcriptase-polymerase chain reaction (RT-PCR), followed byquantitative detection of the product by any of a variety of means;extraction of total RNA from the biological sample, which is thenlabeled and used to probe cDNAs or oligonucleotides encoding all or partof the nucleic acids encoding the proteins/polypeptides of thisinvention, arrayed on any of a variety of surfaces.

The present invention also provides a kit or package comprising reagentsuseful for determining the amount/level of one or moreproteins/polypeptides of the present invention, for example a ligandthat specifically bind to proteins/polypeptides, such as a specificantibody, or to a nucleic acid encoding proteins/polypeptides, such asan oligonucleotide (e.g., primer or probe). Such kit may furthercomprise, for example, instructions for the diagnosis of StaphylococcalIMI, control samples (e.g., samples to which the test sample may becompared to establish the diagnostic), containers, reagents useful forperforming the methods (e.g., buffers, enzymes, immunodetectionreagents, etc). The kit may further include where necessary agents forreducing background interference in a test, agents for increasingsignal, software and algorithms for combining and interpolating markervalues to produce a prediction of clinical outcome of interest,apparatus for conducting a test, calibration curves and charts,standardization curves and charts, and the like. The present inventionalso provides a kit or package comprising one or more agents of thepresent invention for treating and/or preventing Staphyloccocal IMI.Such kit may further comprise, for example, instructions for theprevention and/or treatment of IMI in a mammal.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the followingnon-limiting examples.

EXAMPLE 1 Materials and Methods

Staphylococcus aureus strains. Two types of Staphylococcus aureusisolates from cows were used in this study: chronic and systematicallyisolated strains (i.e. strains isolated from bovine mastitis withclinical signs (high somatic cell counts (SCC) in milk or signs ofinflammation). Chronic isolates were from cows shedding a geneticallyidentical S. aureus strain >55 days apart, between dry off and calvingas illustrated in FIGS. 1 (1^(st) and 2^(nd) samples). Systematicallyisolated strains were taken at calving or in the lactation period fromcows shedding high somatic cell counts (SCC) in milk or signs ofinflammation (mastitis). For the experimental infections describedfurther below, 3 chronic strains were used (#3, #557 and #1290) and werecompared to SHY97-3906, a previously described strain isolated from atypical mastitis case with clinical signs (Diarra et al., 2002) and of aknown in vitro transcriptome (Allard et al., 2006). The collection ofisolates used in this study is shown in Table II below.

TABLE II Staphylococcus aureus mastitis isolates used in the studiesdescribed herein Interval Isolate Type Date (days) Herd Cow Quarter 3Chr 20 Oct. 2005 — 3 37 1 151 Chr 4 Jan. 2006 77 3 37 1 54 Chr 18 Nov.2005 — 2 147 4 353 Chr 10 Feb. 2006 85 2 147 4 140 Chr 1 Jan. 2006 — 846 3 552 Chr 3 Apr. 2006 93 8 46 3 205 Chr 31 Jan. 2006 — 8 40 4 996 Chr20 May 2007 110  8 40 4 557 Chr 3 Apr. 2006 — 3 16 1 1429 Chr 29 Jun.2006 88 3 16 1 2099 Chr 25 Aug. 2006 — 2 96 3 3992 Chr 17 Nov. 2006 85 296 3 1290 Chr 16 Jul. 2006 — 1 83 4 2483 Chr 8 Sep. 2006 55 1 83 4 2484Chr 15 Sep. 2006 — 4 39 1 4210 Chr 8 Dec. 2006 85 4 39 1 3237 Chr 29Sep. 2006 — 4 36 4 4334 Chr 22 Dec. 2006 85 4 36 4 G3  R 15 Mar. 2006 —12 19 C G6  R 16 Mar. 2006 — 11 249 C G7  R 16 Mar. 2006 — 11 156 C G11R 22 Mar. 2006 — 5 28 C G17 R 3 Apr. 2006 — 10 106 C G18 R 24 Mar. 2006— 7 15 C G23 R NA — NA NA NA G26 R 6 Apr. 2006 — 6 135 C G28 R 13 Apr.2006 — 5 32 C G51 R 16 Nov. 2005 — 5 13 C 275 R NA — NA NA NA SHY97-3906R From mastitis; Diarra, MS et al., 2002; Allard et al., 2006 NANewbould R From mastitis; Prasad and Newbould, 1968 (ATCC 29740) NA ATCC49775 — Reference strain from human — ATCC 51811 — Reference strain fromhuman — MRSA COL — Reference MRSA strain from human — N315 — ReferenceMRSA strain from human — Chr, chronic; R, random; C, mix of allquarters; NA, not available

Comparative genomic hybridization of S. aureus Isolates. The geneticrelatedness of S. aureus isolates was determined by comparative genomicDNA hybridization data obtained for 530 genes printed on arrays asdescribed previously (Atalla et al., 2008) and is shown in FIG. 2.

Production of biofilms by S. aureus isolates. Biofilm formation wasevaluated by spectrophotometry in microplates using crystal violetstaining, as previously described with a few modifications (Brouilletteet al., 2005). Briefly, strains were cultured from frozen stocks ontoBHI agar plates and incubated overnight at 35° C. Three colonies werethen inoculated into 7 ml of BHI containing 0.25% of supplementalglucose and incubated at 35° C. for 18 h with shaking at 225 rpm. Thisculture was then diluted to 0.5 McFarland in BHI 0.25% glucose andtransferred into wells of a flat-bottom polystyrene microtiter platehalf full of the same medium. The plates were then incubated at 35° C.for 24 or 48 h. The supernatant was then discarded and the wells weredelicately washed three times with 200 μl of PBS. The plates were dried,stained for 30 min with crystal violet, washed twice with 200 μl ofwater and allowed to dry again. A volume of 200 μl of 95% ethanol wasadded to each well and plates were incubated at room temperature for 1 hwith frequent agitation. The absorbance of each well was then measuredat 560 nm using a plate reader (Bio-Tek Instruments). The results werecollected from at least three independent experiments in which thebiofilm formation of each culture tested was evaluated in fourreplicates.

In vitro culture conditions. For bacterial growth in low and high ironconcentrations, bacteria were first grown in Mueller-Hinton broth (MHB,Becton Dickinson Sparks, Md., USA) in an orbital shaker (225 RPM) at 35°C. At an A_(600 nm) of 0.6 (approx. 1×10⁸ CFU/ml), the culture wasdivided in two pre-warmed sterile flasks. Iron limitation was induced byaddition of 2,2-dipyridyl (Sigma Chemicals, St-Louis, Mo.) at 600 μM toone culture, whereas FeCl₃ was added to the other culture at 10 μM. Thegrowth rate of S. aureus in the presence of supplemental 2,2-dipyridylor FeCl₃ was equivalent in both test conditions and these supplementsdid not affect the exponential growth during the one-hour treatmentperiod. After 1 h, the cultures reached an A_(600 nm) of 1.0 (approx.10⁹ CFU/ml) and 5 ml of each culture were treated with RNAprotect™(QIAgen, Mississauga, ON, Canada) for 10 min before harvesting the cellsby centrifugation. For bacterial growth in freshly collectednon-mastitic milk, S. aureus SHY97-3906, #3, #557 and #1290 were firstgrown overnight in MHB in an orbital shaker (225 RPM) at 35° C. In themorning, 250 ml of fresh milk was inoculated with bacteria from theovernight culture to obtain a bacterial concentration of approximately10⁴ CFU/ml. Bacterial growth was allowed for 7 h in an orbital shaker(225 RPM) at 35° C. before isolating the bacteria from milk as describedbelow. For bacterial growth destined to the qPCR amplification of icaCand hld genes, S. aureus was grown in brain heart infusion (BHI) broth(BD, ON, Canada) until the cultures reached an A_(600 nm) of 0.6.

Animals. All animal experiments were approved by local institutionalanimal care committees and conducted in accordance with the guidelinesof the Canadian Council on Animal Care. Animals were kept in a level 2confinement barn for the entire duration of each trial. EightMultiparous Holstein cows in mid lactation were housed at the Dairy andSwine Research and Development Centre of Agriculture and Agri-FoodCanada in Sherbrooke, QC, Canada. Cows were selected as not infectedbefore the experiment by bacterial analysis of aseptic milk samples andsomatic cell count (SCC) determination.

Experimental infections. Before the animal trials, the relation betweenthe absorbance of bacterial cultures (A_(600 nm)) and CFU was determinedas previously described (Petitclerc et al., 2007). The morning of thechallenge, a volume of the overnight culture of S. aureus in MHB wastransferred to 200 ml of fresh MHB to obtain an A_(600 nm) of 0.1 andgrown at 35° C. without shaking until the A_(600 nm) reached a valuecorresponding to 10⁸ CFU/ml in the exponential phase of growth. Bacteriawere then diluted in sterile physiological saline (Baxter HealthcareCorporation, Deerfield, Ill.) to obtain 50 CFU in 3 ml. Intramammary(IM) infusions were performed the same day immediately after the lateevening milking. Each individual mammary gland quarter was infused with3 ml of a bacterial suspension. Each of the 8 cows was infused with thefour different S. aureus strains and the position of each strain in thefour quarters alternated between the animals. Infusion of mammaryquarters with bacteria was performed according to the proceduredescribed by Nickerson et al. (1999) with few modifications. Allinfusions were performed after milking. Before inoculation, the teat endof each quarter was thoroughly wiped to remove gross contamination anddipped in a solution of iodine. After a minimum of 30 second contacttime, teats were wiped dry and subsequently scrubbed with gauzes soakedin 70% ethanol. Teats were allowed to air-dry. Foremilk was thendiscarded and the IM infusion was performed. Immediately afterwards, allquarters were thoroughly massaged and teats dipped again with an iodinesolution. Disposable gloves were worn throughout the procedure andchanged before proceeding to the next animal.

Milk samples. Milk samples were always aseptically collected beforemilking the experimentally infected cows using the procedure suggestedby the National Mastitis Council (1996). After foremilk was discarded, a10 ml milk sample was collected for each individual quarter in a 50 mlsterile vial. Milk samples were serially diluted and 200 μl plated onTSA and on mannitol salt agar plates (MSA; Becton Dickinson Sparks, Md.,USA) for S. aureus identification. Plates were then incubated for 24 hat 35° C. before colony counting. The dilution that showed between 30and 300 colonies was the one considered for the calculation of bacterialconcentration. Considering the wide range of dilutions plated and thegreat number of samples to be tested, only one plate per sample wasconsidered. Samples that showed 0 colonies for the undiluted milk wasconsidered to have a concentration of 5 CFU/ml. The concentration oflactose, protein, fat and SCC in milk which indicates the presence ofleukocytes in response to an infection were determined in a commerciallaboratory (Valacta Inc., Ste-Anne-de-Bellevue, QC, Canada). This wasdone every two days over the 18-day period of experimental infections.

Milk collection for bacterial isolation. Milk was collected from eachquarter of each cow every 2-4 days in the morning for a total of 18days. Milk was harvested using individual quarter milking units. Priorto milking, the four reservoirs were disinfected with 70% ethanol. Amaximum of one litre of milk was collected for centrifugation andisolation of bacteria.

Bacterial isolation from milk. Mastitic milk from experimentallyinfected cows or freshly collected milk from non-infected cows used forbacterial growth in vitro was treated with 200 μg/ml of protease frombovine pancreas (Sigma) for 10 min in an orbital shaker (100 RPM) at 35°C. After the treatment, the milk was centrifuged 15 minutes at 4000 g.The supernatant was discarded and the pellet was washed with PBS andcentrifuged. The supernatant was discarded and the bacterial cell pelletwas suspended in 1 ml PBS and treated with RNAprotect™ for 10 min beforeharvesting the cells by centrifugation. The cell pellet was then storedfrozen at −86° C.

RNA extraction and purification. Bacterial pellets from in vitro and invivo growth conditions were suspended in 200 μl of TE buffer containing200 μg/ml Lysostaphin™ (Sigma). Cell lysis was allowed for 1 h at roomtemperature before RNA extraction with the TRIzol™ Max bacterial RNAisolation Kit (Invitrogen, Carlsbad, Calif., USA) followed by a DNasetreatment with TURBO™ DNase (Ambion, Austin, Tex., USA). RNA frombacteria isolated from the milk of infected cows underwent an additionalpurification step using the MICROBEnrich™ Kit from Ambion followed by asecond round of DNase treatment with TURBO™ DNase (Ambion). The RNAconcentration in samples was determined by an A_(260 nm) reading and thesamples were stored at −86° C. until used.

cDNA probe synthesis. Fluorescent probes for hybridization to DNA arrayswere generated through an aminoallyl cDNA labelling procedure. Briefly,2.5-5 μg of total RNA was mixed with 5 μg of random hexamers (AmershamBiosciences, Piscataway, N.J., USA). This mixture was denatured at 70°C. for 10 min. Reverse transcription was carried out in the presence ofRT buffer (Invitrogen), 10 mM DDT, dNTP mix (final concentration: 500 μMdATP, dCTP, dGTP, 300 μM dTTP and 200 μM 5-[3-Amioally]-2-dUTP (Sigma))and 400 U of Superscript™ II RT was added to the RNA preparation and thereaction was allowed to occur for 2 h at 42° C. The RNA was hydrolyzedafter transcription with 200 mM NaOH and 100 mM EDTA at 65° C. for 15min. The reaction was neutralized with 333 μM HEPES pH 7.5. The cDNAswere purified before fluorescent labeling through three passages on aMicrocon™ YM30 (Millipore). The resulting aadUTP-cDNA was coupled withNHS-Cy5 (Fluorolink™ Cy5 monoreactive pack, Amersham Biosciences) in thepresence of 100 μM NaHCO3, pH 9.0 for 1 h at room temperature. Thereactions were quenched with 1.25 μM hydroxylamine for 15 min at roomtemperature. The fluorescent cDNAs were purified by using a QIAquick™PCR purification kit (QIAgen), including three washing steps with bufferPE, before eluting in water.

DNA arrays. Arrays were previously described (Allard et al., 2006;Moisan et al., 2006) and contained a selection of 530 known or putativegenes implicated in iron/cation-transport and acquisition systems,virulence (biofilm genes, adhesins, toxins and homologs of such genes),secretion, general stress responses, sensory/regulator systems,antibiotic resistance and various biosynthesis and metabolism genes.Genes were first amplified by PCR using Sigma Genosys™ (Oakville, ON,Canada) primers based on the published genome sequence of the S. aureusCOL genome as well as other primers that were designed using thePrimer3™ software (primer3_www.cgi v 0.2). PCR products were thenpurified using the QIAquick™ PCR purification kit, precipitated,suspended at a concentration of 150 ng/μl in 50% DMSO and printed intriplicate on Corning™ GAPS II slides (Corning, Corning, N.Y., USA) withthe help of the Microarray printing platform of the BiotechnologyResearch Institute of Montreal (Montreal, QC, Canada). Control spotswere from the Lucidea Universal Scorecard (Amersham, Piscataway, N.J.).

Hybridization to DNA arrays and analysis. The probes were suspended in16.5 μl of hybridization buffer (5×SSC, 0.1% SDS, 25% formamide). Theprehybridization, hybridization and washing steps were done asprescribed for Corning™ Gaps II Slides. Hybridization signals for eachspot were quantified with the ScanArrayExpress™ Microarray Scanner andthe ScanArrayExpress™ software V 2.2.0.0022 (Perkin Elmer, Wellesley,Mass., USA). A mean intensity value was calculated as the: Σ (intensityof every spots)/number of genes on array=100%. Only genes with a Cy5signal intensity of ≥100%, i.e., greater or equal to the mean Cy5intensity of the entire array were analyzed. Thus, this reportidentifies only genes that were strongly expressed in vivo duringmastitis because their signal intensities on arrays were higher thanaverage.

Quantitative PCR (qPCR). Additional RNA preparations were obtained forqPCR analyses. Bacteria were collected from broth cultures (low-iron andiron-rich) as well as from milk (in vitro and in vivo) as describedabove. Also, RNA was extracted as mentioned earlier. Total RNA (2-5 μg)was reversed transcribed with 0.5 mM dNTP, 50 ng random hexamers and 200U of Invitrogen Superscript™ II Reverse Transcriptase according to themanufacturer recommendations. RNA was denatured and the cDNAs werepurified with QIAquick™ PCR purification kit. One μl of cDNA wasamplified on the Stratagene™ MX3000P Real-Time PCR (Sratagene, LaJolla,Calif. USA) with a master mix composed of 6 mM Tris-HCl pH 8.3, 25 mMKCl, 4 mM MgCl2, 75 mM trehalose, 0.1% (v/v) Tween™ 20, 0.1 mg/mlnonacetylated BSA, 0.07× SYBR green (Invitrogen), 125 nM dNTPs and 0.5 UJumpStart™ Taq DNA Polymerase (Sigma), and 100 nM of the primers listedin Table III below. Reaction mixtures were denatured for 10 min at 95°C., followed by 40 cycles of 1 min at 60° C., 1 min at 72° C. andfinished with a dissociation ramp from 55° C. to 95° C. The level ofexpression of each gene was calculated by using the Ct of the in vitroexperiments as the calibrator (expression fold=2^(−ΔCt), where ΔCtrepresents the difference between the Ct of the in vitro and in vivoconditions). The fold expression of genes from each experiment was thennormalized with their respective gyrB expression level. The gyrB genewas found to be constitutively expressed during growth up to the earlystationary phase (Goerke et al., 2000), which is well within theboundaries of the growth experiments described herein. Also, it wasfound that the expression of gyrB in the in vitro as well as in the invivo conditions was not significantly modulated.

TABLE III Sequence of primers used for quantitative PCR (qPCR). ForwardReverse ORF Gene Description sequence sequence SACOL0005 gyrBDNA gyrase, B GGTGCTGGGCAAATA TCCCACACTAAATGG subuni CAAGT TGCAA(SEQ ID NO: 1) (SEQ ID NO: 2) SACOL0148 capM Capsular AGGTCCTAGACCAGCTCTCTCCCATCACTT polysaccharide GCTTT GAGC biosynthesis (SEQ ID NO: 3)(SEQ ID NO: 4) SACOL0442 Exotoxin, CATACACAGTTGCTG CAAGCCATAGGAAATputative GCAGAG ATGAGCA (SEQ ID NO: 5) (SEQ ID NO: 6) SACOL0718 ABCGCACAAGAAGTGTTG GTCGTTTTCCCAGAT transporter, CGAGA CCAGA unknown (SEQ ID NO: 7) (SEQ ID NO: 8) function SACOL2022 hld Delta-AATTTAAGGAAGGAG TTTTTAGTGAATTTG hemolysin, TGATTTCAATG TTCACTGTGTCRNA III (SEQ ID NO: 9) (SEQ ID NO: 10) SACOL2171 Unknown CAATGCATCGCGAAAGCTTAGCTTGTGGGA function, ACTTA ACTGG possibly (SEQ ID NO: 11)(SEQ ID NO: 12) iron-related SACOL2325 Transcriptional CATCTCGGCTTAGGTTTTTTCGGCCTAAGT regulator, LysR TACGC TTGGA family (SEQ ID NO: 13)(SEQ ID NO: 14) SACOL269 icaA Biosynthesis of TTGCGTTAGCAAATGAATGCGTGCAAATAC polysaccharides, GAGAC CCAAG biofilms (SEQ ID NO: 15)(SEQ ID NO: 16)

Sequence alignments. Nucleic acid and amino acid sequences of S. aureusgenes (including SACOL0442 and SACOL0720, as well as other genes) andencoded proteins from Staphylococcus aureus strains COL, RF122, NCTC8325, JH1, JH9, Newman, Mu3, Mu50, USA300-FPR3757, N315, MW2 or MSSA476were obtained from the Comprehensive Microbial Resource (CMR) of the J.Craig Venter™ Institute athttp://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi (Peterson, J. D.,et al., Nucleic Acids Res. 2001 29(1): 123-5). The sequences weresubmitted to a multiple sequence alignment program for DNA or proteins,ClustalW2™, available online for free from the European BioinformaticsInstitute (www.ebi.ac.uk; Larkin M. A. et al., 2007. Bioinformatics23(21): 2947-2948).

Purification of proteins encoded by S. aureus genes expressed duringIMI. Genes or part of the genes were cloned into the vector pQE-30(Qiagen) downstream to a polyhistidine signal to allow proteinexpression in Escherichia coli and purification of the expressedhis-tagged polypeptides using a nickel affinity column (Qiagen Ni-NTA1018244). Expression of the recombinant proteins and their purificationwas performed according to the manufacturer's recommendations (Qiagen).

Immunization of mammalian species and measurement of antibody titers.Mice were immunized with the antigens (purified recombinant proteins,polypeptides or epitopes of interest, alone or in combination). Forexample, each animal group composed of ten mice received a differentantigen (100 μg per injection), a combination of antigens (100 μg ofeach per injection) or saline (i.e. the control non-immunized group).Mice were immunized twice 3 weeks apart. The antigens or saline wascombined with the adjuvant Emulsigen®-D (MVP Technologies, Omaha, USA).Injections were performed subcutaneously in 400 μl on the back of themice. Blood samples were performed in the mandibular vein before eachinjection and, 3 weeks after the second injection, mice were euthanizedand maximum blood was sampled. The levels of specific antibodies againstthe immunizing antigens were determined. Levels of antibodies wereevaluated using standard ELISA methodology (Loiselle et al., 2009).Briefly 96-well plates were coated with individual purified antigen andthen saturated with non-specific protein. After incubation with serialdilutions of the serums and washes, a secondary antibody conjugated toan enzyme (HRP) was added and the presence of antibodies was detectedwith a colorimetric reaction.

Immunization in cows. Each animal group composed of 5 cows receives adifferent antigen (300 μg per injection), a combination of antigens (300μg of each per injection) or saline (i.e. the control non-immunizedgroup). The antigens or saline is combined with the adjuvantEmulsigen®-D. After blood samplings for the determination of pre-immunelevels of antibodies, a final volume of 3 ml per dose of antigens orsaline is injected subcutaneously in the neck of the cows. Bloodsamplings is performed every 2 weeks. Ten weeks after the firstinjection, the second injection is performed subcutaneously in the neckon the other side of the animals. The levels of the specific antibodiesis determined as described for the mice immunization.

Evaluation of antibody binding on bacterial surface. Bacteria wereincubated at 4° C., under gentle agitation, with a solution of PBS-2%BSA containing a 1/500 dilution of rabbit serum to block staphylococcalprotein A, which can bind non specifically the Fc fragment ofimmunoglobulins. After 2 washes with PBS-2% BSA-0.02% Tween20™, bacteriawere incubated at 4° C., under gentle agitation, in PBS-2% PBScontaining 10 μl of bovine pre immune or immune serum against theantigen of interest. After 2 washes with PBS-2% BSA-0.02% Tween20™,bacteria were incubated for one hour at 4° C., under gentle agitation,in PBS-2% PBS containing a 1/1000 dilution of FITC-conjugated goatanti-bovine IgG. After 3 washes with PBS-2% BSA-0.02% Tween20™, bacteriawere suspended in PBS with 1% formaldehyde. Surface labeling was thenanalyzed by flow cytometry using a BD FACSCalibur™ instrument and theCellQuest™ Pro software.

Identification of B cell epitopes. With a combination of predictionsoftware including BCPred Predictions (EL-Manzalawy et al., 2008a), AAPPredictions (Chen J et al., 2007), FBCPred Prediction (EL-Manzalawy etal., 2008b) and ABCPred (Saha, S. and Raghava G. P. S., 2006), availableat http://bioinfo.bgu.ac.il/bsu/immunology/epitope_pred/index.htm,http://ailab.cs.iastate.edu/bcpreds/index.html and elsewhere, B cellepitopes, i.e., short amino acid sequences that will be recognized by Bcells, thus inducing the production of antibodies by B cells, weredetermined for several vaccine components.

Identification of T cell epitopes. Computer driven algorithms can alsobe used to facilitate identification of T cell epitopes i.e., shortamino acid sequences that will bind MHC molecules (MHC class I and/orII) and be recognized by T cells, thus inducing a cellular immuneresponse. The antigens may be subjected to analysis by the Epimatrix™System (http://www.epivax.com/platform/) to identify putative T cellepitopes. This in-silico technique divides the total sequence of theantigen into fragments of 9 amino acids overlapping by 8 amino acids.This pool of 9-mer peptides is then screened for predicted affinityagainst a group of known MHC class I and class II alleles. The resultingscores can be used to rate putative epitopes on a common scale which canthen be tested in vitro. The technique is applicable to any animal forwhich a sufficient knowledge of MHC sequences is available. (De Groot etal., 2008).

EXAMPLE 2 Validation of Chronic S. aureus Strains

Comparative genomic hybridization data for the members of chronicisolate pairs collected from cows >55 days apart between dry-off andcalving (FIG. 2, underlined isolates), show a high genetic relatedness.Unrelated reference strains (S. aureus N315, MRSACOL, Newbould, ATCC49775, ATCC 51811 and SHY97-3906) and isolates randomly orsystematically picked from bovine mastitis cases with clinical symptomsduring lactation (annoted “R” in Table I above) are also shown in FIG. 2for comparison. This analysis confirmed that the isolates that werecollected from the same cow and the same quarter >55 days apart, weregenetically identical. It is clear that the chronic isolates #3, #557,and #1290 used in the studies described herein have the ability to causean IMI and persist in the mammary gland for a long period of time (i.e.,are able to cause a chronic IMI).

FIG. 3 shows Q-PCR analyses reporting the relative level of geneexpression for indicators of virulence such as hld (Agr-dependentexotoxin production), icaC (ica-dependent biofilm production) andoverall biofilm production (measured by a spectrophotometric method withcrystal violet) for S. aureus isolates grown in a cultivation medium invitro. Chronic isolates (#3, 557, 1290) were compared to a collection ofsystematically isolated strains from bovine mastitis with clinical signs(where isolate SHY97-3906 is represented as the open square). Q-PCRresults are presented as fold-expression relative to the referencestrain Newbould (ATCC 29740) and biofilm production is reported as apercentage of that produced by strain SHY97-3906. All Q-PCR results werenormalized based on the level of expression of gyrA. The primers usedfor the analysis are shown in Table II above. This analysis confirmedthat the chronic isolates used in the Examples described hereinsubstantially differ in their basal level of gene expression for knownvirulence determinants and for biofilm production compared with thepopulation of systematically collected isolates from clinical mastitiscases during lactation. These characteristics described for the chronicisolates resemble those reported for S. aureus strains isolated frompersistent bovine IMI (Melchior et al., 2009).

EXAMPLE 3 Efficient Isolation of Bacteria from the Milk ofExperimentally Infected Cows

The method used for isolating bacteria from mastitis milk samples isillustrated in FIG. 4. The bacterial pellet recovered from milk treatedwith proteases (prot+, FIG. 4) was much larger and allowed greateramounts of microbial RNA to be isolated for DNA microarray and qPCRexperiments compared to that obtained from the untreated bacterialpellet (prot−, FIG. 4). A DNA microarray experiment comparing thetranscriptional profiles of S. aureus grown in vitro in milk treatedwith casein protease to that of S. aureus cells grown in untreated milkdid not show significant gene modulation. Quantitative PCR analysis for4 genes expressed in S. aureus grown in an iron-restricted medium invitro and under iron-rich conditions show that a 2-hour delay before RNAextraction (time period between milking and RNA extraction) did notaffect the observed modulations in expression of iron-regulated genes(isdB, ferritin and SACOL2170) and did not affect expression of thehousekeeping gene gyrB. The integrity of bacterial messenger RNAdirectly isolated from mastitis milk should therefore not be affected bythe time required for isolation of bacteria after milking of theinfected cow.

EXAMPLE 4 Experimental Infection Profiles in Cows

Experimental infection profiles for strain SHY97-3906 and the 3 chronicstrains (#3, 557, 1290) in 3 different cows are reported in FIGS. 5A-5Cas a function of bacterial (left Y axis) or somatic cell counts (right Yaxis) over the infection period. Data show that all bacterial isolatesare able to establish an intra-mammary infection in cows although thehost (cow) seems to influence the level of bacterial counts, cow #313showing low bacterial counts vs. cow #5325 showing high counts for alltested isolates. Milk samples with high bacterial counts (obtained fromcows #307 and 5325) were thereafter used for transcriptional analyses.

EXAMPLE 5 S. aureus Genes Expressed During IMI in Cows

The transcriptional profile of S. aureus strains infecting the mammaryglands of cows was determined by DNA microarray experiments. Therelative levels of expression of the differentially expressed genes andthe 20 genes expressed by both of the two groups of isolates (i.e., fromchronic or acute mastitis) are reported in Table IV below. FIG. 6 showsthe Venn diagram of the genes differentially expressed in the chronicstrains taken all together (isolates #3, #557 and #1290) versusSHY97-3906 isolated from a typical mastitis case with clinical signs(acute mastitis). This analysis shows that the two types of isolates(chronic vs. typical mastitis isolate (acute)) present different geneexpression profiles and that specific genes may be more stronglyexpressed in each group. These specific sub-groups of genes constitutetherapeutic or vaccine targets to treat specific clinical cases. Also ofinterest are the genes commonly expressed by both types of isolates (20genes in this case, identified by a plus [+] sign in Table IV below),which may be used to treat acute and/or chronic cases.

FIG. 7 shows the Venn diagram of the 43 genes found to be stronglyexpressed in microarray experiments (Table IV below) using bacterialsamples from cow #307 at day 8 (A) and day 10 (B) of infection, and incow #5325 at day 10 of infection (C). The number of bacterial samples inwhich the genes were shown to be expressed is indicated in parenthesisand the gene names that are represented in bold characters were chosenfor qPCR analyses (FIG. 8). This analysis allows identification of genesthat are expressed by one or more isolates in one or more cows at one ormore time points during the infection.

Several genes shown in FIG. 7 were thus expressed in one of thefollowing situations: (i) expressed in more than one strain, (ii)observed in more than one cow and/or (iii) at more than one time point.The expression of 5 such interesting S. aureus genes in 5 to 12independent samples collected from cows with IMI was thus verified andconfirmed by qPCR (FIG. 8). These included the capsular biosynthesisgene (cap), a gene of unknown function (SACOL2171), a transcriptionalregulator of unknown function (SACOL2325), an ABC transporter of unknownfunction (SACOL0718) and a chromosomally encoded gene not previouslycharacterized (SACOL0442). In parallel, gene expression was compared tothat measured in S. aureus cultivated in vitro in Muller-Hinton brothsupplemented with iron (broth+iron), in iron-restricted broth(broth−iron), and in freshly collected non-mastitic milk in vitro (milkin vitro) in order to identify the environmental stimuli involved ingene expression (FIG. 8).

It was observed (i) that the expression of capM was reduced in cows andin milk compared to that seen in vitro, (ii) that gene SACOL2171 wasup-regulated by iron restriction either in cows, in milk or iniron-restricted broth in vitro, (iii) that the expression of SACOL0718and SACOL2325 were specifically induced by the milk environment (i.e.up-regulated in cows compared to any broth in vitro but equivalent tothat seen in fresh milk) and (iv) that gene SACOL0442 was exclusivelyexpressed during infection in the cow, i.e., more expressed in cowscompared to any other environment. The summary of the expression profiledetermined by DNA array and qPCR analyses for genes SACOL0442 andSACOL0718 in different strains, cows and time points during infection isreported in Table V below. As seen, SACOL442 and SACOL0718 arerepresentative examples of S. aureus genes that exhibit sustainedexpression during IMI and this independently of individual S. aureusstrains. Table VI below lists 11 genes (i.e. SACOL442, SACOL0718 and 9other genes) for which expression had never been reported before, whenS. aureus was grown in “other” mammalian environments (i.e. differentfrom the bovine mammary gland environment, as used herein) (Allard etal., 2006; Burlak et al., 2007; Goerke et al., 2000; Garzoni et al.,2007) or in surrogate cultivation media such as in human neutrophils invitro (Voyich et al., 2005), an iron-restricted medium in vitro (Allardet al., 2006; Maresso et al., 2006), in milk in vitro (Lammers et al.,2000) or when S. aureus mastitis isolates were grown in vitro (Tavernaet al., 2007). The genes depicted in Table VI thus represent excellenttargets for prevention and/or treatment of S. aureus IMI, for example ascomponents for a vaccine composition aimed at preventing S. aureus IMI.Also, reports of S. aureus genes expressed in surrogate media or inmammalian environment other than the mammary gland can actually leadaway from what is reported here for S. aureus genes expressed duringbovine IMI. For example, the gene capM (SACOL0148) was reported to beexpressed in a mastitis isolate grown on a blood agar plate in vitro(Taverna et al., 2007) but is shown here to be less expressed duringbovine IMI than that measured after growth in vitro (FIG. 8). It hasbeen previously demonstrated that the genes capM, csb33, csb28, pflB,glpK, and SACOL0154 (listed in Table III above) were all less expressedduring growth of S. aureus in tissue cages implanted in the peritonealcavity of mice than when measured in vitro (Allard et al., 2006),whereas a strong expression of all these genes during bovine IMI isshown in the instant studies. Indeed, the host defense barriers andimmune response, the infected mammary gland tissue and tissue damage, aswell as the altered composition and low oxygen tension of the mastitismilk of cows suffering from IMI all create a unique and complexenvironment that would be difficult to mimic in other animal models ofinfection, in surrogate systems or other cultivation media (Mayer etal., 1988; Park et al., 2007).

Gene SACOL0718 identified in Tables III and V is part of an operoncomprising genes SACOL0718-SACOL0720 as illustrated in FIG. 9 and asdetermined by programs known by those in the field (Prediction ofoperon: www.microbesonline.org, Dehal P. S. et al., Nucleic Acids Res.2010 January; 38(Database issue): D396-400. Epub 2009 Nov. 11; Promotersearch: www.softberry.com, Srivastava S et al., Bioinformation 2008;3(4):173-6. Epub 2008 Dec. 6). The predicted function of these genes isthe formation of an ABC transporter composed of an ATP-binding proteinand a permease (Table V below). SACOL0720 was not detected in themicroarray experiments (Table III above) because it was not included inthe composition of the DNA array (Allard et al., 2006). However, it iswell known that genes from operons are expressed from the same promotersequence and are translated into proteins from the same messenger RNA.Therefore, given that expression of SACOL0718 is detected during IMI, itmay be predicted that expression of SACOL0720 certainly also occurs andthus that both SACOL0718 and SACOL720 represent targets for preventionand/or treatment of S. aureus IMI.

Table IV: S. aureus genes (43 genes) with significant levels ofexpression (intensity>100%) during bovine IMI as determined inmicroarray experiments. Genes are listed by name (if attributed) as wellas by open reading frame (ORF) numbers for three different S. aureusstrains for which the genome is sequenced (MRSA COL, N315 and themastitis isolate RF122). Such genes are also reported in the Venndiagrams of FIGS. 6 and 7. The 20 genes expressed by both chronicstrains as well as by strain SHY97-3906 (common) are indicated by a plus(±) sign.

TABLE IV Cow 307, ORF ORF ORF Cow 307, Day 8 Day 10 Gene COL RF122 N315Description Common SHY97 #3 #557 #1290 SHY97 0029 — 35 Biosynthesis ofcofactors + 106.6 sbnA 0100  55 112 Staphylobactin biosynthesis 440.5sbnB 0101  56 113 Staphylobactin biosynthesis 559.4 sbnF 0105  60 117Staphylobactin biosynthesis 395.7 capM 0148 102 156 Capsularpolysaccharide biosynthesis + 1187.2 291.6 592.3 568.2 177.9 0154 108162 Aldehyde dehydrogenase pflB 0204 164 218 Formate acetyltransferase +pflA 0205 165 219 Formate-lyase activating enzyme + 0264  216c 266 ABCtransporter, unknown function 0442 321 357 Exotoxin, putative + 383.8115.3 201.3 123.5 guaA 0461 341 376 GMP synthase + 396.3 213.0 720.5555.2 331.7 sdrC 0608 513 519 Virulence adhesin + 132.3 adh 0660 557 562Alcool deshydrogenase, Zn containing + 110.5 mntC 0688  581c 587Manganese ABC transporter mntA 0690  583c 589 Manganese ABC transporterfhuA 0704 596 602 Ferrichrome transport ATP-binding protein 0718 610 616ABC transporter, unknown function + 116.8 171.5 108.7 trxB 0829 717 719Thioredoxin reductase menB 1054 912 898 Enoyl-CoA hydratase/isomerasefamily + 132.2 115.0 isdD 1142 996 979 Iron transport from heme + srtB1145 999 982 Sortase B + 107.4 glpK 1320 1161  1141 Glycerol kinase 1353— 1157 ABC transporter, unknown function 115.5 1416 1236c 1213 ABCtransporter, unknown function 103.9 1611 1426c 1383 Transcriptionregulator homolog 118.0 dnaK 1637 1452c 1409 Chaperone protein csb8 1680— 1452 Conserved protein 120.9 isdH 1781 1590c 1552 Iron transport fromheme + 120.1 rot 1812 1622c 1583 Regulator of toxin, Rot 116.2 splC 18671671c 1629 Serine protease csb33 1912 1788c 1671 Glucosamine-6-phosphateisomerase + 110.8 186.8 1944 1818c 1702 Hypothetical protein + murA 20921984c 1902 UDP-NAcGlc-1-carboxyvinyltransferase + 104.2 2144 2033c 1958ABC transporter, unknown function 377.6 2169 2060c 1981 Siderophorebiosynthesis, putative + 2171 2062  1983 Siderophore biosynthesis,putative + 100.4 114.0 144.0 csb28 2321 2205c 2119 Oxidoreductasedehydrogenase/reductase 2325 2209  2123 Transcriptional regulator, LysRfamily + corA 2342 2226c 2137 Magnesium and cobalt transport protein106.9 2365 2248c 2158 Hypothetical protein + 106.9 csb19 2379 2261  2170Conserved protein 116.2 2385 2266  2175 HSP20 family protein 123.7 25992457c 2369 Homolog to FeoB, Fe2+ transport protein Proportion of genes(%) with significant level of expression (intensity >100%) on arrays 6.65.4 7.5 5.9 16.7 Cow 307, ORF ORF ORF Day 10 Cow 5325, Day 10 Gene COLRF122 N315 Description #3 #557 #1290 SHY97 #3 0029 — 35 Biosynthesis ofcofactors 195.4 356.4 sbnA 0100  55 112 Staphylobactin biosynthesis sbnB0101  56 113 Staphylobactin biosynthesis sbnF 0105  60 117Staphylobactin biosynthesis capM 0148 102 156 Capsular polysaccharidebiosynthesis 347.7 181.0 208.7 351.9 0154 108 162 Aldehyde dehydrogenase136.8 pflB 0204 164 218 Formate acetyltransferase 1825.5 465.6 pflA 0205165 219 Formate-lyase activating enzyme 1300.8 231.8 0264  216c 266 ABCtransporter, unknown function 111.8 0442 321 357 Exotoxin, putative227.2 145.1 115.8 guaA 0461 341 376 GMP synthase 335.6 135.6 156.5 209.8sdrC 0608 513 519 Virulence adhesin 173.6 312.1 adh 0660 557 562 Alcooldeshydrogenase, Zn containing 2228.3 168.2 mntC 0688  581c 587 ManganeseABC transporter 273.8 mntA 0690  583c 589 Manganese ABC transporter168.8 fhuA 0704 596 602 Ferrichrome transport ATP-binding protein 162.10718 610 616 ABC transporter, unknown function 110.6 trxB 0829 717 719Thioredoxin reductase 258.3 menB 1054 912 898 Enoyl-CoAhydratase/isomerase family 112.8 222.6 isdD 1142 996 979 Iron transportfrom heme 142.5 200.0 srtB 1145 999 982 Sortase B 109.3 125.2 434.1672.1 glpK 1320 1161  1141 Glycerol kinase 163.2 1353 — 1157 ABCtransporter, unknown function 1416 1236c 1213 ABC transporter, unknownfunction 117.1 1611 1426c 1383 Transcription regulator homolog dnaK 16371452c 1409 Chaperone protein 264.1 csb8 1680 — 1452 Conserved proteinisdH 1781 1590c 1552 Iron transport from heme 117.2 rot 1812 1622c 1583Regulator of toxin, Rot splC 1867 1671c 1629 Serine protease 111.0 csb331912 1788c 1671 Glucosamine-6-phosphate isomerase 176.5 158.2 220.9 19441818c 1702 Hypothetical protein 138.0 277.4 murA 2092 1984c 1902UDP-NAcGlc-1-carboxyvinyltransferase 116.4 2144 2033c 1958 ABCtransporter, unknown function 2169 2060c 1981 Siderophore biosynthesis,putative 112.8 177.3 2171 2062  1983 Siderophore biosynthesis, putative128.4 csb28 2321 2205c 2119 Oxidoreductase dehydrogenase/reductase 129.92325 2209  2123 Transcriptional regulator, LysR family 242.1 364.9 corA2342 2226c 2137 Magnesium and cobalt transport protein 2365 2248c 2158Hypothetical protein 124.7 198.0 csb19 2379 2261  2170 Conserved protein2385 2266  2175 HSP20 family protein 2599 2457c 2369 Homolog to FeoB,Fe2+ transport protein 101.8 Proportion of genes (%) with significantlevel of expression (intensity >100%) on arrays 16.7 16.1 15.4 7.6 8.3

TABLE V Mastitic milk samples in which the expression of SACOL0442(upper panel) or SACOL0718 (lower panel) was detected on DNA array or byqPCR for 4 different S. aureus strains at 3 different time points in twocows. S. aureus strains Cow Day of infection SHY97-3609 3 557 1290 GeneSACOL0442 307 8 ● ● ● ● 307 10 ● ND ● ● 307 14 ● ● ● ● 5325  10 ● ● ● ●Gene SACOL0718 307 8 ● ● ● ● 307 10 ● ● ND ND 307 14 ● ● ● ● 5325  10 ●● ● ● ND, not detected.

TABLE VI Names and annotations for a selection of 11 genes or operonstaken from the 43 genes found to be strongly expressed in microarrayexperiments (Table III above) and for which expression had never beenreported when S. aureus was grown in a different mammalian environmentor in surrogate cultivation media such as in human neutrophils in vitro,in iron-restricted media or in milk in vitro or when S. aureus mastitisisolates were grown in vitro. Annotations are compared forrepresentatives of the S. aureus sequenced genomes (MRSA COL, N315,RF122 [a mastitis isolate], USA300, MSSA476). Gene SACOL COL N315 RF122USA300 MSSA476 0442 enterotoxin, similar to hypothetical enterotoxin,putative putative exotoxin 2 protein putative exported protein 0718-0720ABC ABC ABC ABC putative ABC transporter, transporter, transporter,transporter, transporter ATP-binding ATP-binding ATP-binding ATP-bindingprotein and protein and protein and protein and protein and permeasepermease permease permease permease 2365 lipoprotein, hypotheticallipoprotein, lipoprotein, lipoprotein, putative protein, similarputative putative putative to TpgX protein 0029 HMG-CoA probable —conserved — synthase, HMG-CoA hypothetical truncation synthase protein1416 peptide ABC oligopeptide probable peptide ABC putative transporter,transporter oligopeptide transporter, oligopeptide permease membranemembrane permease transport system protein, permease permease proteinpermease putative domain (opp2c) 1944 conserved conserved conservedconserved putative hypothetical hypothetical hypothetical hypotheticalmembrane protein protein protein protein protein 1611 transcriptionalferric uptake zinc-specific ferric uptake zinc-specific regulator, Furregulator metalloregulator regulation metalloregulatory family homolog(zur) protein (fur) protein 2599 conserved hypothetical probabletransporter putative domain protein protein, similar membrane gatedomain membrane to ferrous iron protein protein protein transporter 2144ABC ABC ABC ABC ABC transporter, transporter, transporter, transporter,transporter, ATP-binding ATP-binding ATP-binding ATP-binding ATP-bindingprotein protein protein protein protein 1353 ABC hypothetical — ABCputative transporter, protein, similar transporter, membrane permease toABC permease protein protein, transporter protein putative integral 0264ABC conserved probable ABC ABC putative ABC transporter, hypotheticaltransporter ATP transporter, transporter ATP- ATP-binding proteinbinding protein ATP-binding binding protein protein protein

EXAMPLE 6 Attenuation of S. aureus Virulence

Mutants for genes SACOL0442 and SACOL0720 were produced by genereplacement for mutant Δ442a, (Mitchell et al., 2008) and by introninsertion for mutants Δ442b and Δ720 (TargeTron Gene Knockout System,Sigma Aldrich (Chen et al., 2007)). The mutants were carried out in theS. aureus parental strain ATCC 29213 that could be easily transformed byelectroporation. For creating mutant Δ442a, a 223-pb fragment of geneSACOL0442 in strain ATCC 29213 was deleted and replaced by insertion ofthe 1300-bp erythromycin resistance gene ermA between positions 188 and411 of the nucleotide sequence of SACOL0442. For creating mutants Δ442band Δ720, the Group II intron (fragment size of approx. 2 Kb) from theTargeTron Gene Knockout System inserted itself into the targetchromosomal gene between nucleotide positions 45 and 46 for geneSACOL0442 and between positions 803 and 804 for gene SACOL0720,respectively. Prior to experimental IMI with the mutants, their growth,compared to the parental strain, was evaluated in vitro in freshlycollected milk (FIG. 10A). No difference between the growth of the 3mutants and the parental strain was observed. Eight healthy lactatingcows were then inoculated intramammary with 25-250 CFU of the parentalstrain and of the three mutants and the infection was followed for 21days. Each of the 8 cows was infused with the four S. aureus strains andthe position of each strain in the four quarters alternated between theanimals. Milk of the infected quarters was collected and thedetermination of viable bacterial counts was performed. Each of thethree mutants showed a significant reduction of bacterial counts in milkcompared to the parental strain (FIG. 10B). The virulence of each of themutants is thus attenuated compared to the parental strain. Theseresults demonstrate the importance of the expression of genes SACOL0442and SACOL0718-720 for the infection process. Antibodies directed towardtheir gene products should greatly impair the ability of S. aureus tocause bovine IMI (see Examples 8-11). Besides, attenuated bacterialstrains have been used as live vaccines (for example, PRIORIX® is acombined measles, mumps and rubella, live, attenuated vaccine;VARILRIX®, is a varicella virus vaccine, live, attenuated (Oka-strain);BCG, is a vaccine for tuberculosis using the attenuated live bacteriaMycobacterium bovis) and thus, the use of the mutants described herein(e.g., Δ442a, Δ442b and Δ720 mutants) for immunization of cows isanother approach to stimulate immunity and to protect the animal againsta future infection by a fully virulent strain. Hence, amutation/deletion of any of the genes identified here as being expressedby S. aureus during bovine IMI may attenuate virulence and suchresulting attenuated mutants could be used in a live attenuated vaccinemethod for immunization.

EXAMPLE 7 Relatedness of Some S. aureus Genes and Proteins

FIGS. 11A-11D show the nucleic acid (FIGS. 11A-11C) and amino acid (FIG.11D) alignments of vaccine components SACOL0442 and SACOL0720 of allStaphylococcus aureus sequenced strains, including the strain RF122isolated from bovine mastitis. The sequences for SACOL0442 and SACOL0720show a similarity of about 94 to 100% among the compared strains and arethus considered as highly conserved among these representative S. aureusstrains. The fact that these genes/proteins could be found in strainsisolated from multiple sources strengthens their potential as targets(e.g., vaccine candidates) as it would target most S. aureus udderinfections (IMI infections).

Similarly, Table VII below shows the percentage of similarity andidentity of the amino acid sequences corresponding to some of the S.aureus genes expressed in vivo during bovine IMI (Table VI above) forsome representatives of the sequenced S. aureus genomes. Again, a highdegree of similarity and identity was observed (>92.7%), confirming thatthese genes and encoded proteins represent good target for protectionagainst multiple S. aureus strains. There is also about 40% identity andabout 60% similarity between the amino acid sequence of SACOL0442 andthat of other putative exotoxins such as SACOL0469, SACOL0470, SACOL0472and SACOL0473 (also known as SA0383 exotoxin 7 [set7], SA0384 exotoxin 8[set8], SA0385 exotoxin 9 [set9] and SA0389 exotoxin 13 [set13] instrain N315, respectively) (www.jcvi.org). Although these components arenot the same genes or proteins, it is possible to find common proteinregions, fragments or epitopes for use in vaccines with broaderapplications and thus aim at the prevention and control of many types ofS. aureus infections in addition to IMI. Some genetically relatedbacterial species or genus such as Staphylococcus epidermidis,Streptococcocus, Listeria and others may also have homologs of thesegenes or proteins. Thus it may also be possible to find common proteinregions, fragments or epitopes for use in vaccines with broaderapplications aimed at the prevention and control of many types ofbacterial infections. For example, the S. aureus gene SACOL1416 showsabout 30% sequence homology to Streptococcus agalactiae gene SAJ1496 andListeria gene LWE0119 and the S. aureus gene SACOL0718 shows about40-50% sequence homologies to Streptococcus agalactiae gene SAJ1013 andListeria gene LWE1764 (www.jcvi.org). Noteworthy, Streptococcusagalactiae is also a pathogen involved in IMI and Listeria is a pathogenoften contaminating milk products (Bradley, 2002; Jayarao et al., 2001).

TABLE VII Percentage similarity (% sim) and identity (% ide) of theamino acid sequences corresponding to some of the S. aureus genesexpressed in vivo during bovine IMI (Table IV above) for somerepresentatives of the sequenced Staphylococcus aureus genomes (strainsN315, RF122, USA300-FPR3757 and MSSA476 compared to the MRSA COLstrain). Gene COL N315 RF122 USA300 MSSA476 SACOL % ide % sim % ide %sim % ide % sim % ide % sim % ide % sim 0442 100 100 99.5 99.5 94.6 98100 100 95.6 98.0 0718 100 100 99.6 100 99.6 100 99.6 100 99.6 100 0720100 100 99.4 99.7 99.4 100 100 100 99.4 99.7 2365 100 100 98.5 98.5 97.198.1 100 100 99.0 99.0 0029 100 100 100 100 — — 100 100 — — 1416 100 10099.6 100 98.2 99.6 100 100 100 100 1944 100 100 100 100 99.6 99.6 100100 100 100 1611 100 100 100 100 100 100 100 100 100 100 2599 100 10099.8 100 99.1 99.8 100 100 99.8 99.8 2144 100 100 94.6 98.5 92.7 96.299.6 99.6 96.2 99.2 1353 100 100 99.6 99.6 — — 100 100 99.6 100 0264 100100 99.5 99.5 99.1 99.5 100 100 99.1 99.1

EXAMPLE 8 Preparation of Vaccines

Bioinformatic software provided sequence and structural information onproteins SACOL0718, SACOL0720 and SACOL0442 that were useful forpreparing such proteins in vaccine compositions (FIGS. 14A-C). Forexample, protein SACOL0442 was determined to be extracellular (secretedbacterial protein). The cellular localization of protein SACOL0718 andits amino acid composition showing 45% of hydrophobic amino acidssuggest that it is associated with the bacterial cytoplasmic membrane.Protein SACOL0720 was also predicted to be associated with the bacterialcytoplasmic membrane. Protein SACOL0720 contains 10 transmembranehelices and it was possible to identify a region exposed at the surfaceof the bacterium.

For vaccine preparation, most of the SACOL0442 protein was used(polypeptide comprising amino acids 44 to 159 in the sequence depictedat FIG. 11D (the full sequence of SACOL0442 is set forth in SEQ ID NO:37)) and as such, excluded its transport signal (amino acids 1 to 35).The predicted extracellular region of protein SACOL0720 (o-annotatedamino acids 309 to 508 in the sequence depicted at FIG. 14D (the fullsequence of SACOL0720 is set forth in SEQ ID NO: 62)) was also used in avaccine composition. In the same way, the extracellular region of theSACOL1781 protein (polypeptide comprising amino acids 41 to 895 ofprotein IsdH (the full sequence of SACOL1781 is set forth in SEQ ID NO:76), see also FIG. 18); (www.jcvi.org)) was used as an additionalvaccine component.

EXAMPLE 9 Immunogenicity of Vaccine of the Present Invention in Mice

Each of the purified polypeptides derived from SACOL0442, SACOL0720 andSACOL1781, independently or all together in combination, were tested forantibody production in mice. Antibody titers in sera of mice vaccinatedwith SACOL0442, SACOL0720 and SACOL1781 (in the presence of the adjuvantEmulsigen®-D) are shown in FIG. 15. One group of animals (10 animals pergroup) twice received saline, one received 2 injections of 100 μg ofpolypeptide SACOL0442, one group received 2 injections of 100 μg ofpolypeptide SACOL0720, one group received 2 injections of 100 μg ofpolypeptide SACOL1781, and one group received 2 injections of 100 μg ofeach three polypeptides SACOL0442, SACOL0720 and SACOL1781 premixedtogether in a combination. The 2 injections were performed 3 weeksapart, and 3 weeks after the second immunization mice were euthanizedand blood collected for the determination of antibody titers by ELISA.Results from FIG. 15 show that the polypeptides used for immunizationwere indeed immunogenic (i.e., able to stimulate an immune response andantibody production). Results also show that the combination ofpolypeptides SACOL0442 and SACOL0720 to another antigen such asSACOL1781 did not reduce or alter antibody production compared to thatmeasured when SACOL0442 and SACOL0720 were injected independently. Sucha vaccine composition (SACOL0442 and/or SACOL0720 with or without otherantigens) is thus a practical useful approach for raising antibodiesagainst multiple antigens of interest. Such a combination vaccine couldthen provide protection against bovine IMI as well as protection againstother diseases that may require other vaccine components forimmunization.

EXAMPLE 10 Immunogenicity of Vaccine of the Present Invention in Cows

Immunizations were also performed in dairy cows. Antibody titers in seraof cows vaccinated with the polypeptide fragments of SACOL0442,SACOL0720 described in Example 8 (in the presence of the adjuvantEmulsigen®-D) are shown in FIG. 16. One group of animals (5 animals pergroup) received 2 injections of saline, one group received 2 injectionsof 300 μg of polypeptide SACOL0442, one group received 2 injections of300 μg of polypeptide SACOL0720 and one group received 2 injections of300 μg of each of the two polypeptides SACOL0442 and SACOL0720 premixedtogether in a combination. The 2 injections were performed 10 weeksapart, and blood was collected for the determination of total IgGantibody titers by ELISA. Results from FIG. 16 show that thepolypeptides used for immunization were also highly immunogenic in cowsand that combining the antigens for immunization also does notsignificantly modify the immune response compared to that obtained usingindividual antigens. The determination of the isotypes is presented inFIGS. 17A-D. Immunization of cows leads to the induction of an immuneresponse with the presence of both IgG1 and IgG2. Isotype IgG2 is knownto be helpful for opsonization of S. aureus and to increase bovineneutrophil functions (Guidry et al., 1993; Barrio et al., 2003). It isknown that using different types of adjuvant and/or vaccineadministration vehicles or routes can modulate the resulting balance ofIgG1 and IgG2 for specific needs (Spickler and Roth, 2003). The capacityof bovine antibodies induced by immunization to bind their targetproteins (e.g., SACOL0720) at the bacterial surface was evaluated.Bacteria grown for 8 hours in freshly collected milk were used for thisassay as this condition was shown to allow expression of SACOL0718-720as measured by qPCR (FIG. 8). Evaluation of antibody binding on thebacterial surface was done using flow cytometry as described in “Example1 materials and methods”. It was found that 22.2% more bacteria werebound by labeled antibodies in the presence of the bovine immune serumraised against SACOL0720 in comparison to the labeling obtained inpresence of the control pre-immune serum. This demonstrates that bovineantibodies induced against SACOL0720 are able to bind to the protein atthe surface of the bacteria. Such antibody binding (opsonization) isknown to help neutrophils phagocytic and killing activity (Guidry etal., 1993; Barrio et al., 2003).

EXAMPLE 11 Epitopes of Interest

As an alternative of using the entire proteins or a long region of thepolypeptides of interest for vaccination, it is also possible tospecifically used small peptide regions predicted to be recognized bythe B or T cells from the mammalian immune system. Identification of theB cell epitopes (that is to say short amino acid sequences that will berecognized by the immune system and able to induce the production ofantibodies by the B cells) among some of the proteins of interest suchas SACOL0442 and SACOL0720 are shown in Table VIII below. For eachprotein, the predicted B cell epitopes are presented with their positionin the protein sequence. The score was obtained from 4 distinctprograms: BCPred Predictions, AAP Predictions, FBCPred Predictions andABCPred.

Similarly, computer driven algorithms can also be used to facilitate theidentification of T cell epitopes (that is to say short amino acidsequences that will be recognized by the immune system and able toinduce a cellular response by T cells) for use as vaccines againstStaphylococcus aureus infection. The proteins of interest can besubjected to analysis by the Epimatrix™ system to identify putative Tcell epitopes. This in-silico technique divides the total sequence ofthe antigen into fragments of 9 amino acids overlapping by 8 aminoacids. This pool of 9-mer is screened for predicted affinity against agroup of known MHC class I and class II alleles. The resulting scorescan be used to rate putative epitopes on a common scale which can thenbe tested in vitro. The technique is applicable to any animal for whicha sufficient knowledge of MHC sequences is available. (De Groot et al.,2008)

The B or T cell epitopes can therefore be used in vaccine compositionsalone or in combination with an assemblage of proteins, peptides orother epitopes. In addition, any B or T cell epitopes as well as anyother epitopes can be presented in a contiguous sequence (such as in aprotein fusion approach) by using genetic and protein engineeringmethods.

TABLE VIII Identification of B cell epitopes among some of the proteins of interest. (A) SACOL0442 and(B) SACOL0720. For each protein, the predictedB cell epitopes are presented with theirposition in the protein sequence and theprediction score they obtained using 4 distinctsoftwares: BCPred Predictions, AAP Predictions,FBCPred Predictions and ABCPred. Potential B Position into cell epitopethe sequence score (A) SACOL0442 TFGIYPKADASTQN  26 0.840(SEQ ID NO: 17) KDTINGKSNKSRNW  72 0.848 (SEQ ID NO: 18) KDGGKYTLESHKELQ159 1.000 (SEQ ID NO: 19) (B) SACOL0720 QFGFDLKHKKDALA 468 0.981(SEQ ID NO: 20) TIKDQQKANQLAS 325 0.898 (SEQ ID NO: 21) KDINKIYFMTDVDL428 0.890 (SEQ ID NO: 22) DVDLGGPTFVLND 436 0.993 (SEQ ID NO: 23)

EXAMPLE 12 Use of S. aureus Genes Expressed during IMI as DiagnosticTools

The diagnosis of S. aureus IMI is difficult and requires time.Traditionally, milk samples are taken and shipped to a microbiologylaboratory where cultivation of S. aureus is achieved using variousartificial growth media. Following growth and if growth occur (usually24 h after sample arrival), the microorganism need to be identified asS. aureus among other possible pathogens by a variety of biochemicaltests which could take up an additional 24 h. For milk producers, thisdelay represents a serious economic loss as cows suspected to haveacquired an IMI need to be removed from the milk production herd whilecows not tested for S. aureus but that have subclinical IMI may continueto contaminate the bulk milk tank. It would thus be highly desirable todevelop a novel tool for rapid detection of S. aureus in milk to permita rapid intervention by milk producers or veterinarians.

As an alternative of using traditional microbial cultures to identify S.aureus in milk samples of cows with or without clinical signs of IMI andmastitis, the products of the S. aureus genes identified as expressedduring IMI (either the messenger RNA, the protein or the metabolicproduct subsequent to the protein activity) may be used as diagnostictools. Indeed, the detection of such specific products, for example inmilk, blood or biopsies, would indicate the presence of S. aureus. Sincesuch products are strongly expressed during IMI, their detection wouldalso strongly correlate with this specific type of infection.

For example, detection of the putative exotoxin SACOL0442 that issecreted in the extracellular milieu, i.e., in milk during mastitis,would be a strong indication that the cow is infected by S. aureus sincethe gene is only expressed during IMI. The detection of the putativeexotoxin SACOL0442 can be easily achieved by the use of a specificantibody and an ELISA technique or a dip stick approach or the like andthe signal of detection can be easily amplified by a variety of signalamplification techniques. Such techniques could rapidly be performed bythe microbiology laboratory or even on-farm by the milk producerhimself, hence gaining valuable time. Alternatively, detection ofmessenger RNA (mRNA) from the genes expressed during IMI would alsoindicate the presence of S. aureus in milk. Detection of mRNA ispossible after its release from bacteria by a cell lysis step, copyingmRNA into complementary DNA by reverse transcription and by PCRamplification.

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The invention claimed is:
 1. A pharmaceutical composition comprising:(A) at least one polypeptide that is: (a) a polypeptide encoded bySACOL1867, the polypeptide consisting of SEQ ID NO: 100; (b) apolypeptide comprising an amino acid sequence, wherein the amino acidsequence is at least 80% identical to the full length of the polypeptideof (a); or (c) a polypeptide comprising an immunogenic fragment of thepolypeptide of (a), the fragment consisting of at least 100 consecutiveamino acids of SEQ ID NO: 100; wherein said at least one polypeptide hasthe ability to elicit an immune response in a mammal; and (B) anadjuvant.
 2. The pharmaceutical composition of claim 1, comprising acombination of polypeptides.
 3. The pharmaceutical composition of claim2, wherein said combination of polypeptides further comprises (C),wherein (C) is: (i) (a) a polypeptide encoded by SACOL0442 comprisingresidues 36 to 203 of the amino acid sequence of SEQ ID NO: 37 or SEQ IDNO: 48; (b) a polypeptide comprising an amino acid sequence, the aminoacid sequence being at least 95% identical to the full length of thepolypeptide of (a), and comprising one or more of the following aminoacid sequences: KDTINGKSNKSRNW (SEQ ID NO: 18), KDGGKYTLESHKELQ (SEQ IDNO: 19), XXTINGKSNKXRNW (SEQ ID NO: 101) and KXGGKYTLESHKELQ (SEQ ID NO:102), wherein X is any amino acid residue; (c) a polypeptide comprisingan immunogenic fragment of (a), and comprising one or more of thefollowing amino acid sequences: KDTINGKSNKSRNW (SEQ ID NO: 18),KDGGKYTLESHKELQ (SEQ ID NO: 19), XXTINGKSNKXRNW (SEQ ID NO: 101) andKXGGKYTLESHKELQ (SEQ ID NO: 102), wherein X is any amino acid residue;or (d) any combination of (a) to (c), wherein said polypeptide has theability to elicit an immune response in a mammal; and/or (ii) (a) apolypeptide encoded by SACOL0720 comprising residues 310 to 508 of theamino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 74; (b) polypeptidecomprising an amino acid sequence, the amino acid sequence being atleast 95% identical to the full length of the polypeptide of (a), andcomprising one or more of the following amino acid sequences:QFGFDLKHKKDALA (SEQ ID NO: 20), TIKDQQKANQLAS (SEQ ID NO: 21),KDINKIYFMTDVDL (SEQ ID NO: 22) or DVDLGGPTFVLND (SEQ ID NO: 23); (c) apolypeptide comprising an immunogenic fragment of (a), and comprisingone or more of the following amino acid sequences: QFGFDLKHKKDALA (SEQID NO: 20), TIKDQQKANQLAS (SEQ ID NO: 21), KDINKIYFMTDVDL (SEQ ID NO:22) or DVDLGGPTFVLND (SEQ ID NO: 23); or (d) any combination of (a) to(c), wherein said polypeptide has the ability to elicit an immuneresponse in a mammal.
 4. The pharmaceutical composition of claim 3,wherein (C) is: (i)(c); and/or (ii)(c).
 5. The pharmaceuticalcomposition of claim 1, wherein said adjuvant comprises alum, an oil,cyclic-diguanosine-5′-monophosphate (c-di-GMP), polyphosphasine orpathogen-associated molecular patterns (PAMPS).
 6. The pharmaceuticalcomposition of claim 5, wherein said PAMPS is unmethylated dinucleotides(CpG) or microbial polysaccharides.
 7. The pharmaceutical composition ofclaim 1, wherein said (A) at least one polypeptide, (B) adjuvant, orboth (A) and (B), are comprised in a pharmaceutical composition.
 8. Thepharmaceutical composition of claim 7, wherein said pharmaceuticalcomposition further comprises one or more pharmaceutically acceptableexcipients.
 9. A kit for immunizing a mammal against a Staphylococcalintramammary infection (IMI), comprising (A) at least one polypeptidethat is: (a) a polypeptide encoded by SACOL1867, the polypeptideconsisting of SEQ ID NO: 100; (b) a polypeptide comprising an amino acidsequence, wherein the amino acid sequence is at least 80% identical tothe full length of the polypeptide of (a); or (c) a polypeptidecomprising an immunogenic fragment of the polypeptide of (a), thefragment consisting of at least 100 consecutive amino acids of SEQ IDNO: 100; wherein said at least one polypeptide has the ability to elicitan immune response in a mammal; and (B) instructions to use the kit forimmunizing a mammal against a Staphylococcal IMI.
 10. The kit of claim9, comprising a combination of polypeptides.
 11. The kit of claim 10,wherein said combination of polypeptides further comprises (C), wherein(C) is: (i) (a) a polypeptide encoded by SACOL0442 comprising residues36 to 203 of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 48;(b) a polypeptide comprising an amino acid sequence, the amino acidsequence being at least 95% identical to the full length of thepolypeptide of (a), and comprising one or more of the following aminoacid sequences: KDTINGKSNKSRNW (SEQ ID NO: 18), KDGGKYTLESHKELQ (SEQ IDNO: 19), XXTINGKSNKXRNW (SEQ ID NO: 101) and KXGGKYTLESHKELQ (SEQ ID NO:102), wherein X is any amino acid residue; (c) a polypeptide comprisingan immunogenic fragment of (a), and comprising one or more of thefollowing amino acid sequences: KDTINGKSNKSRNW (SEQ ID NO: 18),KDGGKYTLESHKELQ (SEQ ID NO: 19), XXTINGKSNKXRNW (SEQ ID NO: 101) andKXGGKYTLESHKELQ (SEQ ID NO: 102), wherein X is any amino acid residue;or (d) any combination of (a) to (c), wherein said polypeptide has theability to elicit an immune response in a mammal; and/or (ii) (a) apolypeptide encoded by SACOL0720 comprising residues 310 to 508 of theamino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 74; (b) polypeptidecomprising an amino acid sequence, the amino acid sequence being atleast 95% identical to the full length of the polypeptide of (a), andcomprising one or more of the following amino acid sequences:QFGFDLKHKKDALA (SEQ ID NO: 20), TIKDQQKANQLAS (SEQ ID NO: 21),KDINKIYFMTDVDL (SEQ ID NO: 22) or DVDLGGPTFVLND (SEQ ID NO: 23); (c) apolypeptide comprising an immunogenic fragment of (a), and comprisingone or more of the following amino acid sequences: QFGFDLKHKKDALA (SEQID NO: 20), TIKDQQKANQLAS (SEQ ID NO: 21), KDINKIYFMTDVDL (SEQ ID NO:22) or DVDLGGPTFVLND (SEQ ID NO: 23); or (d) any combination of (a) to(c), wherein said polypeptide has the ability to elicit an immuneresponse in a mammal.
 12. The kit of claim 11, wherein (C) is (i) (c)and/or (ii) (c).
 13. The kit of claim 9, further comprising (D) anadjuvant.
 14. The kit of claim 13, wherein said adjuvant comprises alum,an oil, cyclic-diguanosine-5′-monophosphate (c-di-GMP), polyphosphasineor pathogen-associated molecular patterns (PAMPS).
 15. The kit of claim14, wherein said PAMPS is unmethylated dinucleotides (CpG) or microbialpolysaccharides.
 16. The kit of claim 13, wherein said (A) at least onepolypeptide, (D) adjuvant, or both (A) and (D), are comprised in apharmaceutical composition.
 17. The kit of claim 16, wherein saidpharmaceutical composition further comprises one or morepharmaceutically acceptable excipients.
 18. The pharmaceuticalcomposition of claim 1, wherein said at least one polypeptide (A) is(c).
 19. The kit of claim 9, wherein said at least one polypeptide (A)is (c).
 20. The pharmaceutical composition of claim 9, wherein in (b),the amino acid sequence is at least 90% identical to the full length of(i).
 21. The pharmaceutical composition of claim 1, wherein saidadjuvant comprises unmethylated dinucleotides (CpG).
 22. The kit ofclaim 1, wherein in (b), the amino acid sequence is at least 90%identical to the full length of (i).
 23. The kit of claim 13, whereinsaid adjuvant comprises unmethylated dinucleotides (CpG).